Anti-baff-r antibody formulations

Abstract

The present application provides a kind of aqueous liquid pharmaceutical preparation, it includes human anti-BAFF-R antibody or its antigen binding portion, it includes high concentration of anti-BAFF-R antibody, with suitable viscosity, the viscosity at room temperature is not higher than 10cp, make ultrafiltration replacement liquid / concentration and filling production smoothly, and can satisfy the requirement of viscosity to subcutaneous injection, expand pre-filled needle injector / injection pen etc. Patient self-administration mode, improve the compliance of patient medication, and have improved bioavailability after subcutaneous administration to subject;Compared with the antibody preparation in the prior art, can not contain surfactant, production process is simpler, and reduce the side effects brought by surfactant in clinic, and have excellent stability, can satisfy the stability in the process of production, storage and transportation.

Description

[0001] Cross-references to related applications

[0002] The amino acid sequence of the anti-BAFF-R antibody described in this application is cited from the authorized patent (application number 202010924738.2) filed by the applicant's company on September 7, 2020 (authorization announcement number: CN114149504B). Technical Field

[0003] This invention belongs to the field of pharmaceutical technology, specifically the biopharmaceutical field, and relates to a formulation of a high-concentration anti-BAFF-R antibody preparation and its clinical application. Background Technology

[0004] As research and development of therapeutic protein drugs continues to deepen and market competition intensifies, differentiated design is gradually becoming a new direction in drug development. The main routes of administration for biologics are intravenous bolus or intravenous infusion (IV), which require a clean administration environment, professionally trained personnel, and significant time costs. In contrast, subcutaneous injection (SC) formulations offer significant advantages in convenience and patient compliance, and are more suitable for patients with vascular occlusion or cardiovascular fragility.

[0005] Differentiated research directions offer new opportunities, but the development of subcutaneous (SC) formulations of protein drugs remains challenging due to their inherent properties. Subcutaneous injections typically deliver volumes less than 2 ml, while the effective therapeutic dose of protein drugs is relatively large. Therefore, there is a corresponding need to develop high-concentration protein formulations to meet clinical dosing requirements.

[0006] High-concentration protein formulations typically exhibit increased viscosity, opalescence, and phase separation, leading to the formation of protein aggregates and gel-like substances. High-concentration protein formulations present greater challenges in manufacturing processes (such as ultrafiltration / concentration of the drug substance, mixing, filtration, and filling of the injection solution), monoclonal antibody quality (protein aggregation and viscosity), analytical methods, and syringe research. For formulation development, the core challenge lies in addressing the three major obstacles of solubility, protein aggregation, and viscosity. Protein aggregation often reduces biological activity and increases the risk of immunogenicity; higher viscosity makes biopharmaceutical production difficult and increases costs, leading to potential quality and safety issues and limiting the use of pre-filled syringes.

[0007] Generally, at higher protein concentrations, the increased interaction between proteins leads to a greater tendency for protein aggregation under the influence of steric effects and van der Waals forces. This results in the formation of soluble aggregates such as dimers and trimers, as well as insoluble aggregate precipitates, which pose challenges to their physicochemical stability.

[0008] In summary, improving the stability and reducing the viscosity of injection solutions are key concerns in the preparation of high-concentration protein formulations, and are also industry challenges that urgently need to be addressed. Summary of the Invention

[0009] The purpose of this invention is to provide a formulation containing a high concentration of anti-BAFF-R antibody, which has good physicochemical stability, meets the requirements for formulation stability during production, storage and transportation, and has low viscosity, which facilitates production operations and clinical use, thus enriching the drug options for the treatment of autoimmune diseases.

[0010] This invention provides a stable anti-BAFF-R antibody formulation, comprising:

[0011] a) Antibody against human B lymphocyte stimulating factor receptor (BAFF-R) at a concentration of about 100 mg / mL to about 160 mg / mL, wherein the antibody comprises a heavy chain variable region (VH) of CDR-H1 having the amino acid sequence of SEQ ID NO:1, CDR-H2 having the amino acid sequence of SEQ ID NO:2, and CDR-H3 having the amino acid sequence of SEQ ID NO:3, and a light chain variable region (VL) of CDR-L1 having the amino acid sequence of SEQ ID NO:4, CDR-L2 having the amino acid sequence of SEQ ID NO:5, and CDR-L3 having the amino acid sequence of SEQ ID NO:6, respectively.

[0012] b) Approximately 10 mM to approximately 30 mM of histidine and / or pharmaceutically acceptable histidine salts.

[0013] c) Sucrose at approximately 120 mM to approximately 180 mM

[0014] d) Arginine and / or pharmaceutically acceptable arginine salts, ranging from approximately 10 mM to approximately 30 mM.

[0015] e) Adjust the pH using dilute hydrochloric acid to approximately 5.5 to approximately 5.9.

[0016] In some embodiments, the stable antibody formulation comprises about 150 mg / ml of anti-BAFF-R antibody, about 20 mM histidine, about 150 mM sucrose, about 20 mM arginine and / or a pharmaceutically acceptable arginine salt, wherein the stable aqueous composition is adjusted to pH about 5.7 with dilute hydrochloric acid.

[0017] In some embodiments, the antibody preparation can be used to treat autoimmune diseases.

[0018] The present invention also provides the use of the aqueous pharmaceutical formulation described in the first aspect in the preparation of a medicament for treating an autoimmune disease in a subject in need of such treatment.

[0019] In some embodiments, the treatment includes subcutaneous administration of a stable aqueous pharmaceutical preparation as disclosed herein to the subject.

[0020] In some embodiments, the composition is packaged in single-dose packages of about 0.5 ml to about 2 ml per dose.

[0021] This article also provides articles comprising containers for containing stable aqueous pharmaceutical formulations disclosed herein.

[0022] Beneficial effects:

[0023] 1) The stable aqueous pharmaceutical formulation of the present invention contains a high concentration of anti-BAFF-R antibody. Compared with antibody formulations in the prior art, it can be surfactant-free, the production process is simpler, and the side effects caused by surfactants in clinical practice are reduced. It also has excellent stability and can meet the stability requirements during production, storage and transportation.

[0024] 2) The high-concentration anti-BAFF-R antibody formulation of the present invention has a suitable viscosity, with a viscosity of no more than 10 cp at room temperature, which facilitates the smooth production of ultrafiltration fluid exchange / concentration and filling, and can meet the viscosity requirements for subcutaneous injection, expand the patient self-administration methods such as pre-filled syringes / injection pens, and improve patient compliance. Attached Figure Description

[0025] Figure 1 Stability data at 25±2℃

[0026] Figure 2 Stability data at 5±3℃

[0027] Figure 3 Pharmacokinetic results of a single subcutaneous injection of cynomolgus monkeys

[0028] Figure 4 Results of rabbit in vitro hemolysis test Detailed Implementation

[0029] It should be understood that although the words "comprising" and "including" appear in various embodiments of this specification, in many cases, the relevant embodiments may also be described using the words "composed of" or "substantially composed of". It should be noted that the term "a" or "an" refers to one or more, for example, "an immunoglobulin molecule" is understood to mean one or more immunoglobulin molecules. Therefore, the terms "a," "an," "one or more," and "at least one" are used interchangeably herein.

[0030] Within any of the ranges described herein, the endpoints of the range are included within that range. However, this specification also includes the same ranges where the lower endpoint and / or upper endpoint are excluded. Further properties and variations of the invention will be apparent to those skilled in the art from the entirety of this application, including the accompanying drawings and detailed description, and all such properties are aspects of the invention. Similarly, the properties of the invention described herein can be combined with other embodiments also intended as aspects of the invention, regardless of whether combinations of properties specifically mentioned above constitute aspects or embodiments of the invention. Furthermore, only such limitations described herein as essential to the invention should be considered as such; variations of the invention lacking limitations that are not described herein as essential are also aspects of the invention.

[0031] I. Terminology Explanation

[0032] To enable those skilled in the art to more clearly understand the inventive content and technical connotation of this invention, the inventors of this invention provide the following explanations of the professional terms, symbols, instruments, equipment, auxiliary materials, reagents, and consumables involved, as well as their abbreviations:

[0033] "Instruments and Equipment": Unless otherwise specified, all are commercially available standard instruments and equipment;

[0034] "Excipients": Unless otherwise specified, all are commercially available conventional pharmaceutical excipients;

[0035] "Reagents and Consumables": Unless otherwise specified, all are commercially available standard reagents and consumables;

[0036] "Anti-BAFF-R antibody": refers to a monoclonal antibody obtained from Chinese hamster ovary (CHO) cells expressing the anti-BAFF-R antibody gene through cell culture, isolation, and purification. Unless otherwise specified, it is manufactured in-house by our company.

[0037] “IV”: Intravenous injection;

[0038] “SC”: subcutaneous injection;

[0039] “DP”: drug product;

[0040] "HMWS": High molecular weight substances;

[0041] “LMWS”: Low molecular weight substances;

[0042] “AGHC”: Non-glycosylated heavy chain;

[0043] “CEX-HPLC”: Cation exchange high performance liquid chromatography.

[0044] “NT”: Not tested.

[0045] “ELISA”: enzyme-linked immunosorbent assay.

[0046] “cp”: centipoise, a unit of viscosity. 1 cp is one-hundredth of a poise. The viscosity of water at 20°C is approximately 1 cp.

[0047] "High concentration antibody products" (HCAPs) typically refer to preparations with a protein antibody concentration of not less than 100 mg / ml.

[0048] II. Analytical Methods and Evaluation Indicators

[0049] The stability evaluation indicators of the injection solution (also known as pharmaceutical preparation (DP)) disclosed in this invention include, but are not limited to: pH, number of subvisible particles, percentage of non-glycosylated heavy chains (AGHC), percentage of high molecular weight substances (HMWS), percentage of low molecular weight substances (LMWS), percentage of total acid peaks, percentage of total basic peaks, protein concentration, percentage of relative binding activity, and percentage of biological activity.

[0050] pH

[0051] Measuring the pH of the DP solution allows confirmation that it is consistent with previous DP batches at release and throughout its shelf life. The stability of DP is defined as having a pH of approximately 5.5, 5.6, 5.7, 5.8, or 5.9. In one embodiment, after storage at approximately 2°C–8°C for approximately 12 months or longer, or after storage at approximately 25°C for approximately 6 months, the pH ranges from approximately 5.5 to approximately 5.9.

[0052] Insoluble particulate analysis

[0053] Based on the average number of subvisible particles, the stability of DP is set as a specific threshold for particulate contamination. The stability of DP is defined as follows: for particles of 10 μm or larger, the average number of particles present in the tested DP cell should not exceed 6000 per container; for particles of 25 μm or larger, the average number of particles present in the tested DP cell should not exceed 600 per container.

[0054] CE-SDS Analysis

[0055] Depending on whether N-ethylcis-butene diimide (NME) or β-mercaptoethanol is used in the sample processing, it is divided into CE-SDS reduction method (using reducing agents such as β-mercaptoethanol and dithiothreitol for sample processing) and CE-SDS non-reduction method.

[0056] Sodium dodecyl sulfate (SDS) is an anionic surfactant that can break the intramolecular and intermolecular hydrogen bonds of protein molecules, disrupt their secondary and tertiary structures, and extend them to maintain similar spatial results. Furthermore, the negative charge carried by the added SDS is much greater than the original charge of the protein. Therefore, its separation is based solely on the molecular weight of the protein, making it a method for determining protein size variants.

[0057] Strong reducing agents such as β-mercaptoethanol and dithiothreitol can break the disulfide bonds between light and heavy chains, as well as between heavy chains, thus separating the light and heavy chains and allowing for the determination of their content.

[0058] The stability of CE-SDS is defined as follows: the sum of the percentages of light and heavy chains in reduced CE-SDS is not less than 95%, and the percentage of non-glycosylated heavy chains (AGHC) is not greater than 5%; the content of monomers in non-reduced CE-SDS is not less than 90%.

[0059] Size exclusion HPLC (SEC-HPLC) analysis

[0060] Size exclusion chromatography (SEC-HPLC), also known as space exclusion chromatography or gel chromatography, is an analytical method that separates solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules. It is mainly used to analyze the size heterogeneity of monoclonal antibody drugs.

[0061] There is no interaction between the sample molecules and the stationary phase. The chromatographic stationary phase is a porous gel that only allows components with a diameter smaller than the pore size to enter. Large molecules in the sample cannot enter the gel pores and are completely excluded. They can only pass through the chromatographic column along the gaps between the porous gel particles and are first eluted from the column by the mobile phase. Medium-sized molecules can enter some suitable pores in the gel, but cannot enter smaller micropores. They are retained in the column and elute from the column more slowly. Small molecules can enter most of the pores in the gel, are more strongly retained in the column, and are eluted even more slowly, thus achieving complete separation of samples with different molecular sizes.

[0062] Monoclonal antibody monomers have a diameter of about 10 nm, while polymers have a larger diameter than monomers and elute before the monomers. Fragments have a smaller diameter than monomers and elute after the monomers. The content of monomers, polymers, and fragments can be calculated based on the order and percentage of peaks to determine the stability of the sample.

[0063] SEC-HPLC can be used to assess the purity of polymeric substances (DPs) and monitor their stability at release and during shelf life. It is an analytical method for examining size variants; a higher monomer percentage indicates better sample quality. DP stability is defined based on various results from SEC-HPLC variables (such as major component (MC), high molecular weight substances (HMWS), or low molecular weight substances (LMWS)). SEC stability is defined as: monomer content not less than 95%.

[0064] Ion chromatography (IEX-HPLC) analysis

[0065] Cation exchange high performance liquid chromatography (CEX-HPLC) belongs to the category of ion exchange chromatography and is mainly used to analyze the charge heterogeneity of monoclonal antibody drugs. Based on the degree of binding with the stationary phase, the substances are eluted at different times. Negatively charged acidic substances are eluted first and rise before the monoclonal antibody, while positively charged basic substances are eluted last and rise after the monoclonal antibody. The content of acidic peaks, monoclonal antibody peaks, and basic peaks can be calculated based on the order and percentage of peaks to determine the stability of the sample.

[0066] CEX-HPLC can be used to assess the purity of charge variants (DPs) and monitor their stability at release and during their shelf life. It is an analytical method for examining charge variants; a higher percentage of the main peak indicates better sample stability. DP stability is defined based on various results from CEX-HPLC variables (such as acid peaks, main peaks, or basic peaks).

[0067] The stability of CEX is defined as follows: the content of the main peak is not less than 40%, the content of the acidic peak is not greater than 40%, and the content of the basic peak is not greater than 20%.

[0068] Relative binding activity

[0069] Enzyme-linked immunosorbent assay (ELISA) is a qualitative and quantitative method that adsorbs soluble antigens or antibodies onto a solid-phase carrier such as polystyrene to perform an immune reaction. It is a major method for detecting binding activity.

[0070] The stability of relative binding activity is defined as 60-140% of that of the reference standard.

[0071] III. Composition of the Formulation

[0072] This invention provides a stable, high-concentration antibody formulation that meets the requirements for formulation stability during production, storage, and transportation, ensures controllable formulation quality, and has a suitable viscosity to meet the needs of production and clinical use.

[0073] 1. Antibodies in the formulation

[0074] In some embodiments, the protein is an anti-BAFF-R antibody, and the concentration of the anti-BAFF-R antibody in the formulation is about 100 mg / ml to 160 mg / ml.

[0075] In some embodiments, the anti-BAFF-R antibody or a fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises CDR-H1, CDR-H2, and CDR-H3 having the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the VL comprises CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0076] In some embodiments, the VH comprises an amino sequence as shown in SEQ ID NO:7.

[0077] In some embodiments, the VL comprises an amino sequence as shown in SEQ ID NO:8.

[0078] 2. Excipients

[0079] Proteins in solution are generally unstable. They can undergo various degradation or aggregation processes, with common degradation pathways including oxidation, deamidation, and isomerization. Excipients are crucial for stabilizing protein formulations during production, storage, and transportation, but their selection is very limited due to regulatory requirements for parenteral formulations.

[0080] In the formulations containing BAFF-R antibodies of the present invention, exemplary excipients include viscosity reducers, freeze-thaw stabilizers, osmotic pressure regulators, and pH buffer systems.

[0081] Typically, the stable formulation composition of high-concentration protein preparations is determined by screening for stable pH ranges and buffer systems, as well as by using stabilizers containing sugars or polyols in the formulation.

[0082] 1) Buffer system and pH range

[0083] The primary structure of an antibody is derived from the dehydration condensation of amino acids. Therefore, antibodies possess amphoteric properties, meaning their charge depends on the pH of their environment. Changing the pH can cause the antibody to carry a positive or negative charge, or it can place it in a zwitterionic state where the number of positive and negative charges is equal (this pH is the antibody's isoelectric point), i.e., a net charge of zero. It is necessary to screen suitable pH and buffer systems to maintain the stability of the antibody solution and meet the pH requirements for subcutaneous injection.

[0084] Most antibodies have an isoelectric point between 8 and 9. Antibodies have the lowest solubility at their isoelectric point, and the pH of the solution should be far from the isoelectric point to maintain antibody stability. The pH of human body fluids is approximately 7.4. To reduce irritation during administration, the pH of the solution for subcutaneous injection is generally between 5 and 8. To maintain solution stability and meet clinical compliance requirements, the pH range of antibody preparations is typically between 5 and 7.

[0085] In some exemplary embodiments, the buffer solution contained in the anti-BAFF-R antibody formulation of the present invention is a buffer solution with a pH of 5.5 to 5.9.

[0086] In some exemplary embodiments, the buffer solution with pH 5.5 to 5.9 is a combination of histidine and dilute hydrochloric acid.

[0087] In some exemplary embodiments, the pH 5.7 buffer solution is a combination of histidine and dilute hydrochloric acid.

[0088] In some embodiments, the histidine concentration in the buffer solution is 10-30 mM.

[0089] In some embodiments, the histidine concentration in the histidine buffer is 18-21 mM.

[0090] 2) Freeze-thaw stabilizers and osmotic pressure regulators

[0091] During the freeze-thaw process, protein formulations are in a concentration and thermodynamic gradient. Monoclonal antibodies close to ice will undergo conformational changes due to adsorption on the ice surface. Sugars, on the other hand, do not crystallize during cryopreservation and can therefore be used as freeze-thaw protectants for monoclonal antibodies.

[0092] Biological membranes, such as human cell membranes or capillary walls, generally possess the properties of semipermeable membranes. The phenomenon of solvent diffusion from a low concentration solution to a high concentration solution through a semipermeable membrane is called osmosis, and the pressure required to prevent osmosis is called osmotic pressure. Osmotic pressure plays a crucial role in various biological processes involving solute diffusion or liquid transport across biological membranes. Therefore, osmotic pressure must be carefully considered when preparing pharmaceutical formulations such as injections and ophthalmic liquid preparations. The osmolality of normal human blood ranges from 285 to 310 mOsmol / kg. Appropriate osmotic pressure regulators need to be added to injections to maintain an osmotic pressure between 240 and 360 mOsmol / kg.

[0093] The polyols in monoclonal antibody formulations can act as freeze-thaw protectants and adjust the osmotic pressure to the level of human blood osmotic pressure.

[0094] Commonly used polyols in antibody preparations include mannitol, sucrose, trehalose, and sorbitol.

[0095] In one exemplary embodiment, the polyol that can simultaneously function as a freeze-thaw protectant and an osmotic pressure regulator is sucrose.

[0096] In some embodiments, the sucrose content in the formulation of the present invention is about 120-180 mM.

[0097] In some embodiments, the sucrose content in the formulation of the present invention is about 150 mM.

[0098] 3) Viscosity reducers

[0099] Dynamic viscosity, also known as dynamic viscosity, absolute viscosity, or simple viscosity, is defined as the ratio of stress to strain rate. Numerically, it is equal to the internal friction generated by the fluid interaction between two flat plates with an area of ​​1 square meter and a distance of 1 meter, moving relative to each other at a speed of 1 m / s. Units: cp (centipoise) or Pa·s (Pa·s), 1 cp = 10-1 -3 Pa·s.

[0100] In some embodiments of the present invention, it has been found that adding a relatively low concentration of certain amino acids to the formulation of the selected antibody reduces the viscosity of the formulation containing the anti-BAFF-R antibody described in the present invention.

[0101] In some embodiments, the viscosity of the anti-BAFF-R antibody formulation is measured before and after the addition of amino acids that reduce viscosity. Methods and equipment for measuring viscosity are well known in the art, such as rotational viscometers.

[0102] In some embodiments, the viscosity-reducing amino acid or its salt, including but not limited to arginine or arginine hydrochloride, and proline.

[0103] In one exemplary embodiment, the viscosity reducer for the high-concentration protein formulation used in subcutaneous administration according to the present invention is arginine hydrochloride.

[0104] In some implementations, the addition of a viscosity reducer can effectively reduce the viscosity of high-concentration protein formulations without adversely affecting the stability of the protein formulations.

[0105] In some embodiments, the concentration of the arginine hydrochloride is from about 10 mM to about 30 mM.

[0106] In one exemplary embodiment, the concentration of arginine hydrochloride is 20 mM.

[0107] IV. Manufactured Products

[0108] This article also provides a manufactured article containing the stable aqueous medicine of the present invention.

[0109] In some implementations, the product is a disposable glass vial equipped with a stopper, containing a stable antibody preparation to be administered.

[0110] In some implementations, the stopper is puncturable with a syringe. In some implementations, the vial is sealed.

[0111] In some implementations, the disposable vial is a 2ml disposable glass vial with a 13mm stopper covered by a 13mm aluminum seal.

[0112] In some implementations, the total volume of the stable pharmaceutical formulation ranges from about 0.5 ml to about 2 ml.

[0113] Table 1: Sequence List

[0114]

[0115]

[0116] Example

[0117] To facilitate understanding of the present invention by those skilled in the art, the present invention provides the following specific embodiments to further illustrate the formulation composition of the high-concentration antibody preparation. These embodiments are merely exemplary and do not limit the scope of this disclosure or the appended claims.

[0118] Example 1: Screening study of formulations at different pH values

[0119] The stability of the monoclonal antibody was investigated at pH values ​​between 5.5 and 7.0. The stock solution was diluted to 150 mg / ml with buffer, and then the pH was adjusted to 5.5, 6.0, 6.5, and 7.0 with sodium hydroxide and / or dilute hydrochloric acid, respectively. The purity was then measured after incubation at 25°C for 1, 3, 5, 7, and 14 days. The SEC-HPLC results are shown in Table 2, and the CEX-HPLC results are shown in Table 3.

[0120] As shown in Tables 2 and 3, the stability of high-concentration antibody formulations varies under different pH conditions. Table 2 shows that the SEC-HPLC results indicate a slight decrease in monomer content at pH 5.5-6.5, primarily due to an increase in the content of low molecular weight substances; at pH 7.0, the monomer content decreases significantly, while the contents of both high and low molecular weight substances increase. Table 3 shows that the CEX-HPLC results indicate a decrease in the main peak content at all pH conditions, primarily due to an increase in the acidic peak. Comparatively, the higher the pH, the faster the main peak content decreases; the formulations exhibit better stability at pH 5.5-6.0. Therefore, further development of high-pH formulations is not considered.

[0121] Based on the combined results of SEC-HPLC and CEX-HPLC, further evaluation of stability at pH 5.5 to 6.0 is considered.

[0122] Table 2: SEC-HPLC purity results of different pH test formulations

[0123]

[0124] Table 3: CEX-HPLC purity results of different pH test formulations

[0125]

[0126]

[0127] Example 2: Sucrose concentration range and freeze-thaw study

[0128] During freezing and thawing, the stock solution is in a concentration and thermodynamic gradient. Proteins near ice will undergo conformational changes due to adsorption on the ice surface. Sugars do not crystallize during cryopreservation and can be used as cryoprotectants for monoclonal antibodies. Sucrose is a commonly used sugar in protein formulations.

[0129] This study was conducted to determine the concentration range of sucrose that could stabilize the JH013 monoclonal antibody against freeze-thaw stress. The study also evaluated the protective effect of sucrose on the monoclonal antibody after five cycles of freeze-thaw cycles.

[0130] Four samples were prepared, with sucrose concentrations of 0, 0.12, 0.15, and 0.18 mol / L for each group. Other formulations remained consistent: protein concentration of 150 mg / ml, pH 5.7, and a buffer system of histidine and dilute hydrochloric acid. Samples were aliquoted into 1 ml 2R vials, sealed, and subjected to a freeze-thaw cycle (first at -40°C for 2 days, then at 25°C for 2 days, constituting one cycle). Samples were taken at 0, 1, 2, 3, 4, and 5 freeze-thaw cycles to determine SEC-HPLC purity.

[0131] As shown in Table 4, after 5 freeze-thaw cycles, the monomer content of each sample did not change significantly compared to the initial value of 0. However, the monomer content of the samples without sucrose decreased slightly faster than that of the samples with sucrose. When the sucrose addition was between 0.12 and 0.18 mol / L, there was no significant difference in purity between SEC-HPLC and the added amount. This indicates that adding 0.12-0.18 mol / L sucrose to the formulation can protect the samples from the effects of freezing and thawing.

[0132] Table 4: Results of the freezing / thawing study

[0133]

[0134] Example 3: Investigation of Arginine Hydrochloride Concentration Range and Viscosity

[0135] High-concentration monoclonal antibody solutions typically have high viscosity, which can cause inconvenience to ultrafiltration concentration, filling processes, and clinical administration. Therefore, an experiment was designed to investigate the concentration range of arginine hydrochloride and its effect on viscosity.

[0136] The experiment was designed and divided into 4 groups, with arginine hydrochloride concentrations of 0, 10 mmol / L, 20 mmol / L, and 30 mmol / L, respectively. Other components were the same: protein concentration of 150 mg / ml, histidine concentration of 20 mmol / L, and sucrose concentration of 150 mmol / L. The pH was adjusted to approximately 5.7 with dilute hydrochloric acid. Each group of samples was prepared with approximately 15 ml of the solution. The viscosity of the four groups of samples was measured at room temperature using a rotational viscometer.

[0137] As shown in Table 5, the viscosity of the sample without arginine hydrochloride was around 17 cp. After adding arginine hydrochloride, the viscosity of the sample decreased to about 7 cp. Within the range of arginine hydrochloride concentration of 10 mmol / L-30 mmol / L, the viscosity of the solution remained similar, around 7 cp, which is much lower than the 17 cp of the sample without the viscosity reducer. This indicates that adding arginine hydrochloride to the formulation can effectively control the viscosity of high-concentration protein injections to meet the requirements of ultrafiltration concentration, filling, and clinical administration.

[0138] Table 5: Results of the study on the concentration range and viscosity of arginine hydrochloride

[0139]

[0140] Example 4: Pharmaceutical Formulation and its Primary Packaging Composition

[0141] 1. This article provides the formulation composition of the anti-BAFF-R antibody preparation (Table 6).

[0142] Table 6: Composition of anti-BAFF-R antibody formulation

[0143]

[0144] 2. The primary packaging of the anti-BAFF-R antibody preparation consists of a glass bottle, a rubber stopper, and an aluminum cap seal, as described in Table 7 below.

[0145] Table 7: Primary Packaging Composition

[0146]

[0147]

[0148] Example 5: Stability under oscillation conditions

[0149] Because monoclonal antibodies are bipolar, their hydrophobic domains readily bind to the water-air interface, leading to aggregation. This aggregation is particularly accelerated by oscillations during production and transportation, which further exacerbate the contact at the water-air interface. Additionally, protein adsorption occurs when it comes into contact with the inner wall of the packaging material, resulting in a decrease in protein content. Therefore, an experiment was designed to investigate the stability of the monoclonal antibody formulation under oscillation conditions.

[0150] Samples were prepared as described in Example 4. The samples were divided into 5 equal portions and dispensed into 2ml vials. The vials were then sealed with stoppers and caps and placed on a shaker at a shaking frequency of 200 rpm / min. One sample was taken out at 0, 1, 3, 5 and 7 days to test the purity, activity and protein concentration.

[0151] As shown in Table 8, when the sample prepared according to Example 4 was shaken at 200 rpm / min for 7 days, all test results were consistent with those at zero time, with no change. This indicates that the formulation can effectively prevent gas-liquid aggregation of high-concentration protein preparations under shaking conditions, as well as the adsorption of protein by the packaging material.

[0152] Table 8: Stability results under oscillation conditions

[0153]

[0154] Example 6: Stability of the formulation under light conditions

[0155] A light exposure experiment was designed to investigate the stability of the anti-BAFF-R antibody preparation under light conditions.

[0156] As described in Example 4, a sample was prepared and divided into 5 equal parts. The prepared sample was placed in a light stability test chamber with the light intensity set at 4500 lx ± 500 lx. One sample was taken out at 0, 1, 3, 5 and 7 days to test the purity, activity and protein concentration.

[0157] As shown in Table 9 below, the SEC-HPLC purity results of the samples prepared according to Example 4, after being placed under 4500 lx ± 500 lx light for 0, 1, 3, 5, and 7 days, were consistent with those at time 0, showing no change. With the extension of exposure time under light conditions, the content of the main peak in CEX-HPLC showed a slow decreasing trend, with no significant change in the acid peak, mainly showing a slight increase in the alkali peak. The sum of the light chain and heavy chain contents of reduced CE-SDS after 7 days of light exposure was consistent with those at time 0, showing no change. The monomer content of non-reduced CE-SDS decreased slightly. The binding activity and protein content results at 7 days were consistent with those at time 0, showing no change.

[0158] Table 9: Stability data under illumination conditions

[0159]

[0160] Example 8: Stability of DP under high temperature conditions

[0161] This study was conducted to test the stability of the anti-BAFF-R antibody formulation at a high temperature of 40°C.

[0162] As described in Example 4, the sample was prepared and divided into 4 equal portions, which were then dispensed into 2ml vials and sealed with stoppers and caps. The prepared sample was placed under a high temperature of 40°C, and one sample was taken out at 0, 7, 14 and 28 days to test its purity, activity and protein concentration, and to examine the high temperature stability of the sample.

[0163] As shown in Table 10 below, under high temperature conditions of 40℃, the monomer content of the sample decreased slightly by SEC-HPLC, while the corresponding HMWS and LMWS increased. However, HMWS increased slowly and linearly, while LMWS increased slowly in the first 14 days and then increased rapidly at day 28. The main peak content of CEX-HPLC decreased significantly, and the acid peak and the basic peak increased in different proportions, with the acid peak increasing slightly more. The sum of the heavy chain and light chain content of reduced CE-SDS decreased slowly, while the non-glycosylated heavy chain showed no significant change. The content of non-reduced CE-SDS monomer showed a clear decreasing trend. The binding activity and protein concentration remained the same as at 0, with no change.

[0164] Table 10: Stability data of DP under high temperature conditions

[0165]

[0166] Example 7: Stability Study

[0167] Long-term stability studies can serve as the primary basis for setting product storage conditions and shelf life. Accelerated testing can be used to understand the product's stability under short-term deviations from storage conditions, providing supporting data for determining shelf life and storage conditions.

[0168] This study was conducted to investigate the stability of the anti-BAFF-R monoclonal antibody formulation of the present invention under various environmental conditions. Three batches of samples were prepared and aliquoted into 1ml vials of the 2ml inner packaging material described in Example 4. The vials were then capped and sealed. The three batches of samples were placed under accelerated storage conditions of 25℃±2℃ and the recommended long-term storage conditions of 5℃±3℃. Samples were taken at planned times to test the stability of the samples. The testing protocols are shown in Table 11.

[0169] Table 11: Inspection Time

[0170]

[0171] Stability study results

[0172] The stability results of formulations containing the anti-BAFF-R monoclonal antibody of the present invention under accelerated and long-term conditions are as follows: Figure 1 and Figure 2 As shown. A comprehensive analysis of the results indicates that...

[0173] 1) After being placed under accelerated conditions at 25℃±2℃ for 6 months, the appearance of the formulation was consistent with that at 0, being a pale yellow clear liquid with a slight opalescence; the molar osmotic pressure, pH, protein content, and activity (binding activity and biological activity) were consistent with those at 0, with no change; the monomer content of SEC-HPLC showed a slow decreasing trend; the main peak of CEX-HPLC decreased significantly, while both acid and alkali peaks increased, with the acid peak showing a higher increase; the sum of the light and heavy chain contents of reduced CE-SDS decreased slightly, while the content of non-glycosylated heavy chain was consistent with that at 0; the content of non-reduced CE-SDS monomer decreased significantly.

[0174] 2) After the formulation was placed at 5℃±3℃ for 18 months, except for the slow decrease in the content of reduced CE-SDS monomers, all other test indicators were consistent with those at 0℃, with no significant difference.

[0175] 3) The stability trends of the three batches of anti-BAFF-R monoclonal antibody preparations were consistent under accelerated conditions of 25℃±2℃ and long-term conditions of 5℃±3℃.

[0176] Therefore, it can be seen that the JH013 high-concentration antibody preparation with consistent product quality can be stably produced according to the present invention. After being stored under the specified storage conditions for 18 months, the product quality is consistent with that at 0, and the stability is good. Although the purity decreases slightly under accelerated conditions at 25°C, it can be tolerated for 6 months, and the stability is excellent.

[0177] The high-concentration anti-BAFF-R antibody formulation of the present invention, comprising anti-BAFF-R antibody, arginine salt, sucrose and histidine buffer, is able to resist stress induced by the air / solution interface and stress induced by the solution / surface, without producing protein aggregation, and can meet the needs of production, storage, transportation and clinical application.

[0178] Example 8: Pharmacokinetic Study of Anti-BAFF-R Antibody Preparation Administered Subcutaneously to Cynomolgus Monkeys via Single-Dose Administration

[0179] This study administered anti-BAFF-R antibody preparations to cynomolgus monkeys via single subcutaneous and single intravenous injections. Blood drug concentrations and anti-antibody levels were measured at different time points before and after administration to investigate the pharmacokinetics and immunogenicity of JH013 injection in cynomolgus monkeys within a defined dose range. Twenty-four cynomolgus monkeys were divided into four groups of six (half male and half female): a low-dose (3 mg / kg), a medium-dose (10 mg / kg), a high-dose (30 mg / kg) subcutaneous injection group, and a medium-dose (10 mg / kg) intravenous injection group. Blood samples were collected at different time points before and after administration. Blood drug concentrations were measured using a validated ELISA method. The main pharmacokinetic parameters were calculated using the Phoenix WinNonlin non-compartmental model (NCA), and anti-antibody levels were detected using a validated ELISA method.

[0180] Table 12: Pharmacokinetic Results of Single-Dose Subcutaneous Injection of JH013 Injection in Cynomolgus Monkeys

[0181]

[0182] like Figure 3 As shown, the mean T values ​​of cynomolgus monkeys were as follows: subcutaneous injection of low (3 mg / kg), medium (10 mg / kg), and high dose (30 mg / kg) and intravenous injection of medium (10 mg / kg) dose. 1 / 2 The durations of action were 222.61±120.17h, 250.55±84.21h, 233.18±168.22h, and 293.36±133.22h, respectively, excluding the pharmacokinetic parameter T of the high-dose subcutaneous injection group. 1 / 2 Except for sex differences in Vz, no sex differences were observed in the major pharmacokinetic parameters of the other groups; drug exposure levels in each dose group were positively correlated with the administered dose. At a dose level of 10 mg / kg, subcutaneous injection of JH013 cynomolgus monkeys was performed based on AUC. 0～1680hThe absolute bioavailability was 83.42%.

[0183] Example 9: In vitro hemolysis test of anti-BAFF-R antibody preparation in rabbits

[0184] This experiment used an in vitro hemolysis test (in vitro tube method) to evaluate whether the test substance induced hemolysis or agglutination in rabbit erythrocytes, providing a reference for safe clinical use. Venous blood was collected from healthy New Zealand rabbits and prepared into a 2% erythrocyte suspension. Several clean test tubes were numbered: tubes 1-1, 2-1, 3-1, 4-1, 5-1 and tubes 1-2, 2-2, 3-2, 5-2, 5-2 were the test substance group 1 (37.25 mg / ml) and test substance group 2 (stock solution), respectively; tube 6 was the negative control; tube 7 was the solvent control; and tube 8 was the positive control. After mixing, the tubes were immediately incubated at 37°C. The results were observed and recorded at 15 min, 30 min, 45 min, 1 h, 2 h, and 3 h. After 3 h, the presence of hemolysis or erythrocyte agglutination was determined, and photographs were taken.

[0185] like Figure 4 As shown, no hemolysis or agglutination occurred in the negative control tube, while hemolysis occurred in the positive control tube. Neither the undiluted solution (149 mg / ml, diluted in vitro to 2.98–14.9 mg / ml) nor the 1:3 diluted test substance (37.25 mg / ml, diluted in vitro to 0.75–3.75 mg / ml) showed hemolysis or agglutination within 3 hours in any tube containing 2% erythrocyte suspension from New Zealand rabbits. This indicates that the test substance and its solvent yielded consistent results, meaning they did not cause hemolysis of erythrocytes in New Zealand rabbits.

[0186] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. (such as adjusting the cushioning system, filling volume, packaging material type, etc.) made within the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A stable antibody formulation, wherein the formulation comprises: a) Anti-BAFF-R antibody or fragment thereof at a concentration of approximately 100 mg / ml to approximately 160 mg / ml; b) A pH-adjusted buffer solution of approximately 10 mM to approximately 30 mM; c) Amino acids with viscosity-reducing effects, at concentrations of approximately 10 mM to approximately 30 mM; and d) Polyols with freeze-thaw stabilizing and osmotic pressure regulating effects, ranging from approximately 120 mM to approximately 180 mM. The pH-adjusting buffer can adjust the pH of the antibody preparation to about 5.5-5.9, and the anti-BAFF-R antibody or a fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL). The VH comprises CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, and the VL comprises CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:

6.

2. The antibody preparation of claim 1, wherein the buffer system adjustable to pH 5.5 to 5.9 is histidine and histidine hydrochloride, or a combination of histidine and hydrochloric acid.

3. The antibody preparation of claim 1, wherein the polyol having freeze-thaw protection and osmotic pressure regulation functions is selected from sucrose, mannitol, trehalose, and sorbitol.

4. The antibody preparation of claim 1, wherein the amino acid with the viscosity-reducing effect is selected from arginine, arginine hydrochloride, histidine, and proline.

5. The antibody preparation according to any one of claims 1-4, wherein the buffer solution is a mixture of histidine and dilute hydrochloric acid with pH adjustable to 5.5-5.9, the polyol having freeze-thaw protection and osmotic pressure regulation effects is sucrose, and the amino acid with viscosity-reducing effect is arginine hydrochloride.

6. The antibody preparation according to claim 5, wherein the concentration of the anti-BAFF-R antibody is 150 mg / ml, the pH is about 5.5-5.9, the histidine concentration in the buffer solution is 18-21 mM, the sucrose concentration is 140-160 mM, and the arginine hydrochloride concentration is 18-21 mM.

7. The antibody preparation of claim 6, comprising: a) Approximately 150 mg / ml of anti-BAFF-R antibody or a fragment thereof; b) Approximately 20 mM of histidine; c) Approximately 20 mM arginine hydrochloride; and d) Approximately 150 mM of sucrose, The pH of the formulation is approximately 5.

7.

8. The antibody preparation according to any one of claims 1-7, which may be used to treat autoimmune diseases and / or tumors.

9. A container comprising the antibody preparation as claimed in any one of claims 1-8.

10. The container of claim 9, wherein the container is a vial, an auto-injector, an injection pen, or a pre-filled syringe.

11. Use of the antibody preparation according to any one of claims 1-8 in the preparation of a medicament for treating autoimmune diseases or tumors mediated by abnormally high expression of BAFF-R in subjects of need.

12. The use as claimed in claim 11, wherein the treatment is administered by subcutaneous injection of the antibody preparation of any one of claims 1-8.

13. The use as described in claim 11 or 12, wherein the autoimmune disease comprises or is selected from one of the following: primary Sjögren's syndrome (PSS), systemic lupus erythematosus, lupus nephritis, idiopathic pulmonary fibrosis, rheumatoid arthritis (RA), autoimmune hepatitis, multiple sclerosis, myasthenia gravis, IgA nephropathy, neuromyelitis optica, granulomatous disease with polyangiitis, microangiitis, immune thrombocytopenic purpura, autoimmune hemolytic anemia, thermophilic autoimmune hemolytic anemia, lupus-like syndrome, lupus nephritis, diffuse sclerosis, psoriasis, inflammatory bowel disease, multiple sclerosis, and rheumatoid arthritis. The tumor diseases mentioned therein include the cancer diseases comprising lymphoma, myeloma and leukemia, such as non-Hodgkin lymphoma (B-NHL), chronic lymphocytic leukemia (CLL), primary acute lymphoblastic leukemia (ALL), diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, and multiple myeloma.

14. The use as described in claim 11 or 12, wherein, The autoimmune disease mentioned is primary Sjögren's syndrome (PSS).

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