Stable pharmaceutical formulation of Anti-CD38 antibody and use thereof

Abstract

Provided in the present invention is a subcutaneous administration formulation comprising a CD38 antibody and hyaluronidase. By means of optimizing a prescription, stable preservation of the pharmaceutical formulation can be maintained, thereby facilitating transportation and long-term preservation, being suitable for clinical subcutaneous injection, and improving the medication compliance of patients.

Description

Stable pharmaceutical formulations of anti-CD38 antibodies and their uses Technical Field

[0001] This invention relates to the field of monoclonal antibody drug formulations, and more specifically to a stable drug formulation comprising CD38 monoclonal antibody and hyaluronidase for subcutaneous administration and its use. Background Technology

[0002] Human CD38 is a type II transmembrane glycoprotein (46 kDa) with a structure similar to that of soluble cyclases isolated from California sea snails. It possesses both extracellular cyclase and hydrolase activities. CD38 is expressed on the surface of hematopoietic cells, multiple myeloma cells, other cell types, and tissues, and performs various functions, such as receptor-mediated adhesion, signal transduction, and regulation of cyclase and hydrolase activities.

[0003] Several anti-CD38 antibodies for the treatment of multiple myeloma and other malignant tumors are currently on the market or in clinical trials, including daratumumab, isatuximab, TJ202, HexaBody-CD38, SAR-442085, and CID-103. Among them, daratumumab, developed by Johnson & Johnson, is a CD38 monoclonal antibody that has received FDA Fast Track designation and Breakthrough Therapy designation. One specification (400mg / 20mL) contains the following formulation: 400mg daratumumab, 3.7mg glacial acetic acid, 510mg mannitol, 8mg polysorbate 20, 59.3mg sodium acetate, 70.1mg sodium chloride, water for injection, pH 5.5.

[0004] Since its FDA approval in 2015, daratumum has seen its indications expand continuously, transitioning from a fourth-line to a first-line treatment, resulting in strong market growth. Global sales reached $9.7 billion in 2023. However, daratumum is administered via intravenous infusion, which is time-consuming and causes inconvenience for patients.

[0005] Hyaluronidase is an enzyme that degrades hyaluronic acid (EC3.2.1.35) and reduces the viscosity of hyaluronic acid in the extracellular matrix, thereby increasing tissue permeability. Human rHuPH20 is a recombinant hyaluronidase, described in Chinese patent CN200480011489.7. The enzymatic hydrolysis of subcutaneous hyaluronic acid by rHuPH20 disrupts the three-dimensional structure of the skin interstitium, reducing its density and viscoelasticity. This increases the retention space and diffusion rate of infusions within the subcutaneous interstitium, reduces the compressive force on tissues, and lowers the likelihood of tissue tearing and damage. The addition of hyaluronidase allows for the conversion of drug formulations with a dosage volume greater than 2 ml from intravenous injection to subcutaneous injection, shortening the administration time from several hours to 2-7 minutes, thus facilitating administration for both doctors and patients and significantly improving patient compliance.

[0006] Most approved antibody drug formulations currently on the market contain the surfactants polysorbate 20 or polysorbate 80, both of which contribute to the stability of hyaluronidase in the drug formulation. Antibody drugs are typically expressed using CHO cells (Chinese hamster ovary cells), followed by purification and capture of the antibody protein using a combination of affinity chromatography, anion exchange chromatography, and cation exchange chromatography. Despite these purification methods, host proteins (HCPs) may still remain in the semi-finished and / or finished antibody drugs from CHO cells. Some HCPs possess lipolytic activity, which can degrade polysorbate 20 or 80 in the drug formulation, negatively impacting the stable storage of antibody drug formulations containing hyaluronidase.

[0007] The removal of host proteins (HCPs) is primarily limited by the choice of chromatographic packing material and chromatographic conditions. Maintaining a balance between target protein purity and yield is crucial; finding suitable affinity chromatography conditions that achieve both high antibody purity (i.e., thorough HCP removal) and high yield is challenging. From the perspective of improving monoclonal antibody production efficiency, reducing production costs, and maintaining process stability, a uniform purification process is desirable for all types of antibodies. However, because the heavy chain amino acid sequences of different antibody types are not identical, finding affinity chromatography conditions suitable for multiple antibody types is difficult. This also poses a challenge to the removal and control of host proteins (HCPs), ultimately increasing the production cost of antibody drugs.

[0008] Therefore, it is necessary to develop a more universal and stable antibody drug formulation containing hyaluronidase, avoiding the use of surfactants polysorbate 20 and / or polysorbate 80, so that even if trace amounts of host protein are present (within the pharmacopoeia standard limits), it will not affect the stable storage of the drug formulation. This has great industrial value for simplifying the purification process of antibody protein drugs and reducing drug production costs. Summary of the Invention

[0009] Through extensive formulation preparation process experiments, the inventors have obtained a stable pharmaceutical composition containing CD38 monoclonal antibody and hyaluronidase, but free of surfactants polysorbate 20 and / or polysorbate 80, thus avoiding the adverse effects of trace amounts of host protein on the stable storage of the pharmaceutical composition.

[0010] On one hand, the present invention provides a pharmaceutical composition suitable for subcutaneous injection, comprising:

[0011] CD38 monoclonal antibodies ranging from 10 mg / mL to 180 mg / mL;

[0012] Hyaluronidase ranging from 50 U / ml to 5000 U / ml;

[0013] 1 mM to 50 mM histidine buffer, pH range 5.0-7.0;

[0014] 50 mM to 400 mM of polyhydroxy alcohols or sugars, selected from sorbitol, trehalose or sucrose;

[0015] 0.01% (w / v) to 2% (w / v) of the surfactant poloxamer 188; and

[0016] Methionine, a stabilizer, at concentrations ranging from 0.1 mg / mL to 2.5 mg / mL.

[0017] In another embodiment, the CD38 monoclonal antibody in the pharmaceutical composition of the present invention is selected from daratumumab, isatuximab, TJ202, HexaBody-CD38, SAR-442085, or CID-103, with daratumumab being the preferred CD38 monoclonal antibody (wherein the host protein content is <1 ng / mg). Information regarding CD38 monoclonal antibodies is described in US7829693, WO05 / 103083, WO06 / 125640, WO07 / 042309, WO08 / 047242, and WO14 / 178820, all of which are incorporated herein by reference.

[0018] In another embodiment, the concentration of CD38 monoclonal antibody in the pharmaceutical composition of the present invention is 50 mg / ml-150 mg / ml, preferably 100 mg / ml-150 mg / ml, more preferably 100 mg / ml-130 mg / ml, and most preferably 120 mg / ml.

[0019] In the pharmaceutical composition described in this invention, the addition of hyaluronidase (HA) is crucial. It can degrade hyaluronic acid in skin tissue, improve tissue permeability, facilitate drug diffusion, increase the volume of subcutaneous injection, reduce the concentration of antibody preparations, avoid the instability caused by aggregation of highly concentrated protein preparations, and also avoid the increase in viscosity caused by the interaction between multiple antibody molecules, which could lead to excessive foaming during use and cause denaturation and inactivation of active biological agents.

[0020] The hyaluronidase described in this invention can be selected from animal or human sample derivatives or manufactured using recombinant DNA technology. Hyaluronidase products approved in EU countries include "Dessau," and animal-derived hyaluronidase products approved in the United States include Vitrase™, Hydase™, and Amphadase™, as well as FDA-approved recombinant human hyaluronidase rHuPH20 (HYLENEXTM) or its mutants. The term rHuPH20 mutant refers to a new protein sequence obtained by mutations, substitutions, or deletions of certain amino acid residues based on the rHuPH20 sequence, which exhibits improved properties compared to rHuPH20 in at least one aspect, such as enzyme activity or thermostability.

[0021] In the pharmaceutical composition of this invention, the hyaluronidase is preferably rHuPH20, having a molecular weight of approximately 61 kDa. Its amino acid sequence and manufacturing method can be found in CN200480011489.7, WO2006 / 091871, and US7767429, all of which are incorporated herein by reference. Further relevant literature can be found in Frost, GI, “Recombinant human hyaluronidase (rHuPH20): an enabling platform for subcutaneous drug and fluid administration”, Expert Opinion on Drug Delivery 4:427-440 (2007). Literature on rHuPH20 mutants can be found in WO2013 / 102144, WO2020 / 022791, and WO2020 / 197230, all of which are incorporated herein by reference.

[0022] The enzyme activity of the hyaluronidase (including rHuPH20) described in this invention can be defined in units / ml (U / ml) or in total enzyme activity (U) in a specific formulation. The standard definition of one unit of enzyme activity (U) in this invention is the amount of enzyme catalyzing a predetermined amount of substrate reaction per unit time, for example, one mole or one nanomole of substrate / minute. Techniques for determining the activity of hyaluronidase formulations are known in the art, and the activity of hyaluronidase formulations is generally expressed in USP units or units.

[0023] In another embodiment, the amount of hyaluronidase in the pharmaceutical composition of the present invention is from 100 U / ml to 4000 U / ml, preferably from 500 U / ml to 3000 U / ml, more preferably from 1500 U / ml to 2500 U / ml, and most preferably 2000 U / ml.

[0024] In another embodiment, the pharmaceutical composition of the present invention preferably contains 5 mM to 30 mM of buffer histidine, with a pH range of 5.0-7.0; preferably contains 5 mM to 20 mM of buffer histidine, with a pH range of 5.0-7.0; more preferably contains 5 mM to 10 mM of buffer histidine, with a pH range of 5.0-6.0; and most preferably contains 8 mM of buffer histidine, with a pH of 5.5.

[0025] In another embodiment, the pharmaceutical composition of the present invention contains 100 mM to 300 mM of a polyhydroxy alcohol or sugar selected from sorbitol, trehalose or sucrose; preferably, it contains 150 mM to 300 mM of trehalose or sucrose; preferably, it contains 200 mM to 300 mM of trehalose or sucrose; most preferably, it contains 250 mM of trehalose dihydrate.

[0026] In another embodiment, the pharmaceutical composition of the present invention uses the surfactant poloxamer instead of polysorbate 20 and / or polysorbate 80. Through extensive experimentation, the inventors of this application have discovered that regardless of whether the CD38 monoclonal antibody and recombinant human hyaluronidase were completely removed or contained only trace amounts of host protein before formulation, the pharmaceutical composition containing CD38 monoclonal antibody and hyaluronidase remains stable after using the surfactant poloxamer 188. As shown in Tables 40-43 of the examples, using different amounts of poloxamer 188 has no significant effect on protein purity; the formulations using poloxamer 188 and those using polysorbate 20 surfactant have no significant effect on protein purity. However, in the stability study, the poloxamer 188 content remained essentially unchanged in the formulation using poloxamer 188, while the polysorbate 20 content decreased to varying degrees in the stability study at different temperatures.

[0027] In another embodiment, the pharmaceutical composition of the present invention contains 0.01% (w / v) to 1% (w / v) of the surfactant poloxamer 188; preferably, it contains 0.01% (w / v) to 0.1% (w / v) of the surfactant poloxamer 188; more preferably, it contains 0.02% (w / v) to 0.06% (w / v) of the surfactant poloxamer 188.

[0028] In another embodiment, the pharmaceutical composition of the present invention contains 0.5 mg / mL to 2.5 mg / mL of the stabilizer methionine; preferably, it contains 0.5 mg / mL to 2 mg / mL of the stabilizer methionine; more preferably, it contains 0.5 mg / mL to 1 mg / mL of the stabilizer methionine; and most preferably, it contains 0.9 mg / mL of the stabilizer methionine.

[0029] As a preferred embodiment, the present invention provides a pharmaceutical composition suitable for subcutaneous injection, comprising:

[0030] Daratumumab at concentrations ranging from 10 mg / mL to 180 mg / mL;

[0031] Hyaluronidase ranging from 50 U / ml to 5000 U / ml;

[0032] 1 mM to 50 mM histidine buffer, pH range 5.0-7.0;

[0033] 50 mM to 400 mM of polyhydroxy alcohols or sugars, selected from sorbitol, trehalose or sucrose;

[0034] 0.01% (w / v) to 2% (w / v) of the surfactant poloxamer 188; and

[0035] Methionine, a stabilizer, at concentrations ranging from 0.1 mg / mL to 2.5 mg / mL.

[0036] As a preferred embodiment, the present invention provides a pharmaceutical composition suitable for subcutaneous injection, comprising:

[0037] daratumumab at concentrations ranging from 50 mg / mL to 150 mg / mL;

[0038] Hyaluronidase at concentrations ranging from 500 U / ml to 3000 U / ml;

[0039] 5mM to 30mM histidine buffer, pH range 5.0-7.0;

[0040] 50 mM to 400 mM of polyhydroxy alcohols or sugars, selected from sorbitol, trehalose or sucrose;

[0041] 0.01% (w / v) to 1% (w / v) of the surfactant poloxamer 188; and

[0042] Methionine, a stabilizer, at concentrations ranging from 0.5 mg / mL to 2 mg / mL.

[0043] As a preferred embodiment, the present invention provides a pharmaceutical composition suitable for subcutaneous injection, comprising:

[0044] daratumumab at concentrations of 100 mg / mL to 150 mg / mL;

[0045] Hyaluronidase at concentrations ranging from 500 U / ml to 3000 U / ml;

[0046] 5 mM to 20 mM histidine buffer, pH range 5.0-7.0;

[0047] 100mM to 300mM of polyhydroxy alcohols or sugars, selected from sorbitol, trehalose or sucrose;

[0048] 0.01% (w / v) to 1% (w / v) of the surfactant poloxamer 188; and

[0049] Methionine, a stabilizer, at concentrations ranging from 0.5 mg / mL to 1.5 mg / mL.

[0050] As a preferred embodiment, the present invention provides a pharmaceutical composition suitable for subcutaneous injection, comprising:

[0051] daratumumab at concentrations ranging from 100 mg / mL to 130 mg / mL;

[0052] Hyaluronidase at concentrations ranging from 1500 U / ml to 2500 U / ml;

[0053] 5mM to 10mM histidine buffer, pH range 5.0-6.0;

[0054] 100mM to 300mM of trehalose or sucrose;

[0055] 0.01% (w / v) to 1% (w / v) of the surfactant poloxamer 188; and

[0056] Methionine, a stabilizer, at concentrations ranging from 0.5 mg / mL to 1.0 mg / mL.

[0057] As a preferred embodiment, the present invention provides a pharmaceutical composition suitable for subcutaneous injection, comprising:

[0058] 120 mg / mL daratumumab;

[0059] 2000 U / ml hyaluronidase;

[0060] 8 mM histidine buffer, pH 5.5;

[0061] 250mM trehalose dihydrate;

[0062] Surfactant poloxamer 188 from 0.02% w / v to 0.06% (w / v); and

[0063] Methionine, a stabilizer, at a concentration of 0.9 mg / mL.

[0064] The present invention provides a pharmaceutical composition containing daratumumab suitable for subcutaneous injection, exhibiting surprising storage stability. The pharmaceutical formulation, by adding hyaluronidase, increases the injection volume for safe and comfortable subcutaneous administration. In some embodiments, the administration volume of the daratumumab-containing pharmaceutical composition is 5-20 ml. In other embodiments, the administration volume is 12-18 ml. In the most preferred embodiment, the administration volume is 15 ml.

[0065] Hyaluronidase promotes the systemic delivery of the therapeutic antibody daratumumab via subcutaneous administration by reversibly hydrolyzing acetylated hyaluronic acid. The hydrolysis of acetylated hyaluronic acid in subcutaneous tissue temporarily opens channels in the interstitial space of the subcutaneous tissue. Subcutaneous administration of the pharmaceutical compositions prepared according to the present invention has been observed to improve the dispersion, absorption, and bioavailability of the therapeutic antibody. Large molecules (i.e., greater than 16 kDa) administered subcutaneously are preferentially absorbed into the vascular compartment via draining lymph. Therefore, the rate at which these large molecules are introduced into the systemic circulation is slower compared to intravenous infusion, potentially leading to a decrease in the frequency / intensity of infusion-related reactions. Additionally, reduced pain and decreased swelling at the injection site have been observed after subcutaneous administration.

[0066] Another aspect of the present invention describes a kit comprising one or more formulations packaged in a manner convenient for subject use as described herein. In one embodiment, this kit comprises the formulations described herein packaged in containers such as sealed bottles, containers, single-use or reusable vials, pre-filled syringes, or pre-filled injection devices, optionally having a label affixed to the container or included in the packaging describing the use of the compound or composition in the implementation of the method. In one aspect, the compound or composition is packaged in a unit dosage form. The kit may further include a device suitable for administering the composition according to a specific route of administration. Preferably, the kit includes a label describing the use of the antibody or formulation described herein.

[0067] The CD38 monoclonal antibody drug composition of the present invention, suitable for subcutaneous injection, can induce the killing of CD38-expressing tumor cells through antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), apoptosis, or in vitro regulation of CD38 enzyme activity. The anti-CD38 antibody in the drug composition of the present invention can also mediate antitumor efficacy through its immunomodulatory effect, which is achieved by inducing CD4+. + and CD8 + Cell proliferation, and / or the suppression of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs)-mediated inflammatory responses.

[0068] In another embodiment, the CD38 monoclonal antibody drug composition of the present invention, suitable for subcutaneous injection, can be used to treat multiple myeloma (MM), smoldering multiple myeloma (SMM), glioblastoma, ITP, plasma cell myeloma, lupus erythematosus, lupus nephritis, Waldenstrom macroglobulinemia, prostate cancer, membranous proliferative glomerulonephritis, malignant tumors of primary lymphoid hematopoiesis, RR-T cell lymphoma, hemolytic anemia, relapsed or refractory chronic lymphocytic leukemia, plasmablastic lymphoma, non-small cell lung cancer, and Alzheimer's disease, among other related diseases.

[0069] In another embodiment, the CD38 monoclonal antibody drug composition of the present invention suitable for subcutaneous injection can be administered in combination with a second therapeutic agent or a combination thereof.

[0070] In another embodiment, the second therapeutic agent is an alkylating agent, selected from busulfan, cyclophosphamide, bendamustine, levofloxacin, carboplatin, cisplatin, temozolomide, melphalan, carmustine, cyclohexanenitrosourea, dacarbazine, oxaliplatin, ifosfamide, dichloroethylmethylamine, thiotepa, trabectedin, or streptozotocin.

[0071] In another embodiment, the second therapeutic agent is a proteasome inhibitor selected from bortezomib, carfilzomib, or ixazomib.

[0072] In another embodiment, the second therapeutic agent is a glutamate derivative selected from lenalidomide, thalidomide, or pomalidomide.

[0073] In another embodiment, the second therapeutic agent is a corticosteroid selected from dexamethasone or prednisone.

[0074] In another embodiment, the CD38 monoclonal antibody drug composition of the present invention suitable for subcutaneous injection can be administered simultaneously or sequentially with a second therapeutic agent or in combination thereof.

[0075] In another embodiment, the CD38 monoclonal antibody drug composition of the present invention, suitable for subcutaneous injection, is used in clinical treatment, wherein the CD38 monoclonal antibody in the drug composition is selected from daratumumab, isatuximab, TJ202, HexaBody-CD38, SAR-442085, or CID-103, with daratumumab being the preferred CD38 monoclonal antibody.

[0076] In another embodiment, the daratumumab pharmaceutical composition of the present invention, suitable for subcutaneous administration, can be used in combination with bortezomib, lenalidomide, and dexamethasone for induction and consolidation therapy in newly diagnosed patients suitable for autologous stem cell transplantation.

[0077] In another embodiment, the daratumumab drug composition of the present invention suitable for subcutaneous injection can be used in combination with lenalidomide and dexamethasone (Rd) to treat newly diagnosed MM patients who are not suitable for autologous hematopoietic stem cell transplantation, as well as relapsed or refractory MM patients who have received at least one first-line treatment.

[0078] In another embodiment, the daratumumab pharmaceutical composition of the present invention suitable for subcutaneous injection can be combined with bortezomib and dexamethasone for the treatment of patients with multiple myeloma who have received at least one prior treatment.

[0079] In another embodiment, the daratumumab drug composition of the present invention suitable for subcutaneous administration can be used in combination with bortezomib, melphalan, and prednisone to treat newly diagnosed MM patients who are not suitable for autologous hematopoietic stem cell transplantation.

[0080] In another embodiment, the daratumumab pharmaceutical composition of the present invention suitable for subcutaneous administration can be used in combination with carfilzomib and dexamethasone to treat patients with relapsed or refractory multiple myeloma who have previously received one to three treatments.

[0081] In another embodiment, the daratumumab pharmaceutical composition of the present invention suitable for subcutaneous injection can be combined with pomalidomide and dexamethasone for the treatment of patients with multiple myeloma who have received at least one first-line therapy, including lenalidomide and proteasome inhibitor therapy.

[0082] In another embodiment, the daratumumab pharmaceutical composition of the present invention suitable for subcutaneous administration can be combined with bortezomib, thalidomide and dexamethasone for the treatment of newly diagnosed patients with multiple myeloma who are eligible for autologous hepatocyte transplantation.

[0083] In another embodiment, the daratumumab drug composition of the present invention, suitable for subcutaneous injection, can be used in combination with bortezomib, cyclophosphamide, and dexamethasone to treat newly diagnosed patients with primary light chain amyloidosis.

[0084] In summary, this invention provides a subcutaneous drug delivery formulation comprising CD38 antibody and hyaluronidase, and its use. The formulation prepared by this invention has the following advantages over existing technologies:

[0085] (1) Reduced production costs and simplified purification process: In the current antibody drug purification process, in order to remove HCP with enzymatic activity, it is necessary to optimize the chromatography conditions for different antibody specificities. This poses a huge challenge in balancing high purity and high yield, resulting in complex production processes and high costs. The formulation of this invention fundamentally reduces the stringent requirements for the purification process, significantly simplifies the production steps and reduces costs.

[0086] (2) More versatile formulation platform: Due to the different characteristics of different antibody molecules, it is difficult to use a uniform purification process to efficiently remove HCP. This invention solves the problem by changing the formulation rather than the purification process, thus eliminating the dependence on a specific antibody sequence and forming a universal formulation applicable to a variety of different antibody drugs.

[0087] (3) Better formulation storage stability: The formulation does not contain polysorbate 20 / 80. Even if there are trace amounts (compliant with standards) of HCP remaining in the product, the physical stability of the drug will not be affected by the degradation of the surfactant (such as precipitation or particles), thus greatly ensuring the quality and safety of the product within its shelf life and making it more stable. Detailed Implementation

[0088] The present invention is further illustrated by the following embodiments, but the embodiments should not be construed as limiting the scope of protection of the present invention.

[0089] Example 1: Study on the role of each excipient

[0090] The effects of each excipient were investigated using different subcutaneous formulations containing CD38 antibody and hyaluronidase. Specific formulation designs are shown in Table 1, pH 5.5. The CD38 antibody used in the formulation studies was daratumumab, prepared according to standard biosimilar development procedures.

[0091] Table 1. Composition of different subcutaneous drug delivery formulations

[0092] The stability of each formulation was investigated at different temperatures (40 / 25 / 5℃) and freeze-thaw cycles (freeze-thaw cycle 1: -20℃ for 2 days, then warmed to 5℃ for 2 days, as one cycle; freeze-thaw cycle 2: -80℃ for 2 days, then warmed to 5℃ for 2 days, as one cycle). Sample solutions were taken out at different time points for testing to evaluate the purity of the active ingredient antibody (size exclusion chromatography SEC purity, capillary isoelectric focusing icIEF purity) and the subvisible particles of the protein solution (microfluidic imaging system MFI) to investigate the chemical and physical stability of the antibody.

[0093] Table 2 Purity Results at 40℃

[0094] Table 3 Results of subvisible particles at 40℃

[0095] Table 4 Purity Results at 25℃

[0096] Table 5 Results of subvisible particles at 25℃

[0097] Table 6 Purity Results at 5℃

[0098] Table 7 Results of subvisible particles at 5℃

[0099] Tables 2-7 show that adding polyhydroxy alcohols, sugars (such as sorbitol), or surfactants (such as polysorbate 20) alone has no significant effect on protein purity. Adding methionine slows down the growth rate of high molecular weight proteins. Samples without added surfactants had more particles at day 0 than those with added surfactants. When placed at 40℃ / 25℃ / 5℃, the samples without added surfactants had significantly more particles.

[0100] Table 8 Purity Results under Freeze-Thaw Conditions

[0101] Table 9 Results of subvisible particles under freeze-thaw conditions

[0102] As shown in Tables 8-9, the samples without surfactants had significantly more particles at the initial day 0 than those with surfactants. During the freeze-thaw cycle, the particle count did not increase significantly for any of the samples. Adding surfactants prevents particle formation. There was no significant difference in icIEF purity between the stock solutions with different components within 5 freeze-thaw cycles. The stock solutions without added polyhydroxy alcohols or sugars showed a faster increase in polymer content than those with added polyhydroxy alcohols, and this increase was more pronounced under slow freeze-thaw cycles (-20℃ to 25℃) than under fast freeze-thaw cycles (-80℃ to 25℃). This indicates that polyhydroxy alcohols or sugars protect proteins at ultra-low temperatures.

[0103] In summary, adding polyhydroxy alcohols or sugars can protect proteins during cryopreservation, adding surfactants can significantly reduce the number of subvisible particles in the solution, and adding methionine can further enhance the physical stability of proteins (reducing the growth of high molecular weight proteins).

[0104] The role of methionine in the prescription of Example 2

[0105] Two subcutaneous drug delivery formulations containing CD38 antibody and hyaluronidase were selected. The changes in the samples were studied under light stress. The specific formulation design is shown in Table 10.

[0106] Table 10 Prescriptions containing and without methionine

[0107] Each prescription sample was placed under indoor ambient light and then exposed to strong light in a light box (illuminance 4500 lx ± 500 lx, with a total illuminance not less than 1.2 × 10⁻⁶). 6 lx·hr, near-ultraviolet lamp energy not less than 200W·hr / m 2 Stability was investigated by taking samples at different time points to evaluate the purity of the active ingredient antibody (size exclusion chromatography, capillary isoelectric focusing, and icIEF purity), the subvisible particles in the protein solution (microfluidic imaging system, and methionine content), and to assess the chemical and physical stability of the antibody.

[0108] Table 11 Purity Results under Indoor Temperature and Fluorescent Lamp Conditions

[0109] Table 12 Results of subvisible particles and methionine content under indoor ambient temperature and fluorescent lamp conditions.

[0110] Table 13 Purity Results under Strong Light Irradiation Conditions

[0111] Table 14 Results of subvisible particles and methionine content under strong light irradiation conditions.

[0112] As shown in Tables 11-14, under room temperature fluorescent lamp conditions for 24 hours, the chemical and physical stability of samples with and without added methionine was consistent. Under strong light irradiation, with the extension of light exposure time, the methionine content continuously decreased, and the protein purity decreased significantly, mainly manifested in a significant increase in high molecular weight proteins and a significant increase in acid peaks.

[0113] To investigate which factor is related to methionine content, methionine-containing samples placed under different temperature conditions for 3 months were tested for methionine content. The results are shown in Table 15.

[0114] Table 15 Results of methionine content at different temperatures

[0115] In summary, methionine, as a commonly used antioxidant, can further reduce the content of high molecular weight proteins under conditions of 40 / 25 / 5℃ compared with samples without added methionine, and significantly reduce the growth of high molecular weight proteins under light irradiation.

[0116] Example 3: Screening of different types of sugars or polyhydroxy alcohols, surfactants, and antioxidant protection measures

[0117] Commonly used sugars or polyhydroxy alcohols and surfactants in biopharmaceutical formulations were selected for screening. At the same time, common antioxidant protection measures were screened, and commonly used antioxidants such as methionine, chelating agents (EDTA), and inert gas nitrogen protection were selected (see Table 16).

[0118] Table 16 Screening of different types of sugars or polyhydroxy alcohols, surfactants, and antioxidant protection measures

[0119] The stability of each formulation was investigated at different temperatures (40 / 25 / 5℃). Sample solutions were taken at different time points for testing to evaluate the purity of the active ingredient antibody (size exclusion chromatography SEC purity, capillary isoelectric focusing icIEF purity) and the subvisible particles of the protein solution (microfluidic imaging system MFI). The chemical and physical stability of the antibody was investigated. The results are shown in Tables 17-22.

[0120] Table 17 Purity Results at 40℃

[0121] Table 18 Results of subvisible particles at 40℃

[0122] Table 19 Purity Results at 25℃

[0123] Table 20 Results of subvisible particles at 25℃

[0124] Table 21 Purity Results at 5℃

[0125] Table 22 Results of subvisible particles at 5℃

[0126] Tables 17-22 show that the chemical and physical stability of proteins is not significantly different when using different sugars or polyhydroxy alcohols, such as mannitol, sorbitol, trehalose, and sucrose. Similarly, the chemical and physical stability of proteins is not significantly different when using different surfactants, such as polysorbate 20 and poloxamer 188. When using polysorbate 80, the physical stability of the protein at 5°C is not significantly different from that of samples using polysorbate 20 and poloxamer 188. At 40 / 25°C, the growth of high molecular weight protein is slightly faster, but overall, the physicochemical stability is within a controllable range (the high molecular weight protein content at 40°C for 3M is 4.65%, which is within the 5% control limit for high molecular weight protein in new drugs; other test items, icIEF and MFI, show no significant difference compared to samples using polysorbate 20 and poloxamer 188). The samples with added methionine showed slower growth of high molecular weight proteins compared to those without; the addition of the chelating agent EDTA had a counter-effect on the physical stability of the protein, resulting in faster growth of high molecular weight proteins; the samples under nitrogen protection showed slower growth of high molecular weight proteins compared to those without methionine, but there was no significant difference in growth between the samples with added methionine and those without.

[0127] Example 4: Screening of different types of sugars or polyhydroxy alcohols and antioxidant protection measures

[0128] Commonly used sugars or polyhydroxy alcohols in biopharmaceutical formulations were selected for screening. Common antioxidant protection measures were also screened, including the commonly used antioxidant methionine, chelating agent (EDTA), and inert gas nitrogen protection (see Table 23).

[0129] Table 23 Screening of different types of sugars or polyhydroxy alcohols and antioxidant protection measures

[0130] The stability of each formulation was investigated at different temperatures (40 / 25 / 5℃). Sample solutions were taken at different time points for testing to evaluate the purity of the active ingredient antibody (size exclusion chromatography SEC purity, capillary isoelectric focusing icIEF purity), the subvisible particles of the protein solution (microfluidic imaging system MFI), the polysorbate 20 content, and to investigate the chemical and physical stability of the antibody and the content of functional excipients.

[0131] Table 24 Purity Results at 40℃

[0132] Table 25 Results of subvisible particles at 40℃

[0133] Table 26 Purity Results at 25℃

[0134] Table 27 Results of subvisible particles at 25℃

[0135] Table 28 Purity Results at 5℃

[0136] Table 29 Results of subvisible particles at 5℃

[0137] Tables 24-29 show that using different sugars or polyhydroxy alcohols, such as mannitol, sorbitol, trehalose, and sucrose, does not significantly affect the chemical and physical stability of proteins. Samples with added methionine show slower high-molecular-weight protein growth compared to samples without added methionine. The addition of the chelating agent EDTA has a counter-effect on the physical stability of proteins, resulting in faster high-molecular-weight protein growth. Nitrogen-protected samples show slower high-molecular-weight growth than samples without added methionine, but there is no significant difference in high-molecular-weight growth between samples with added methionine and those with added methionine.

[0138] Table 30 Results of Polysorbate 20 Content

[0139] Table 30 shows that when polysorbate 20 is used as a surfactant, its content decreases during stable storage, exhibiting a significant temperature effect; the lower the temperature, the slower the degradation. After 3 months of high-temperature storage at 40℃ and 6 months of accelerated storage at 25℃, the polysorbate content decreased to 0.008% and 0.010%, respectively. Further reduction may lead to physical instability of the formulation.

[0140] Example 5: Screening of different types of sugars or polyhydroxy alcohols, surfactants, and antioxidant protection measures

[0141] Commonly used sugars or polyhydroxy alcohols and surfactants in biopharmaceutical formulations were selected for screening. At the same time, common antioxidant protection measures were screened, and commonly used antioxidants such as methionine, chelating agents (EDTA), and inert gas nitrogen protection were selected (see Table 31).

[0142] Table 31 Screening of different types of sugars or polyhydroxy alcohols, surfactants, and antioxidant protection measures

[0143] The stability of each formulation was investigated at different temperatures (40 / 25 / 5℃). Sample solutions were taken at different time points for testing to evaluate the purity of the active ingredient MAB (size exclusion chromatography SEC purity, capillary isoelectric focusing icIEF purity), the subvisible particle content of the protein solution (microfluidic imaging system MFI), and the content of polysorbate 20. The chemical and physical stability of MAB and the content of functional excipients were also investigated.

[0144] Table 32 Purity Results at 40℃

[0145] Table 33 Results of subvisible particles at 40℃

[0146] Table 34 Purity Results at 25℃

[0147] Table 35 Results of subvisible particles at 25℃

[0148] Table 36 Purity Results at 5℃

[0149] Table 37 Results of subvisible particles at 5℃

[0150] Tables 32-37 show that the chemical and physical stability of proteins is not significantly different when using different sugars or polyhydroxy alcohols, such as mannitol, sorbitol, trehalose, and sucrose. Similarly, the chemical and physical stability of proteins is not significantly different when using different surfactants, such as polysorbate 20 and poloxamer 188. When using polysorbate 80, the physical stability of the protein at 5°C is not significantly different from that of samples using polysorbate 20 and poloxamer 188. At 40 / 25°C, the growth of high molecular weight protein is slightly faster, but overall, the physicochemical stability is within a controllable range (the high molecular weight protein content at 40°C for 3M is 4.60%, which is within the 5% control limit for high molecular weight protein in new drugs; other test items, icIEF and MFI, show no significant difference compared to samples using polysorbate 20 and poloxamer 188). The samples with added methionine showed slower growth of high molecular weight proteins compared to those without; the addition of the chelating agent EDTA had a counter-effect on the physical stability of the protein, resulting in faster growth of high molecular weight proteins; the samples under nitrogen protection showed slower growth of high molecular weight proteins compared to those without methionine, but there was no significant difference in growth between the samples with added methionine and those without.

[0151] Table 38 Results of Polysorbate 20 Content

[0152] Table 38 shows that when polysorbate 20 is used as a surfactant, its content decreases during stable storage, exhibiting a significant temperature effect; the lower the temperature, the slower the degradation. After 3 months of high-temperature storage at 40℃ and 6 months of accelerated storage at 25℃, the polysorbate content decreased to 0.013% and 0.016%, respectively. Further reduction may lead to physical instability of the formulation.

[0153] Example 6: Screening of Poloxamer 188 Dosage and Comparison of Poloxamer 188-Containing Formulations with Polysorbate 20

[0154] The dosage of poloxamer 188 in the formulation was changed, and commonly used sugars or polyhydroxy alcohols and surfactants in biopharmaceutical formulations were screened. Common antioxidant protection measures were also screened, and commonly used antioxidants such as methionine and inert nitrogen protection were selected (see Table 39).

[0155] Table 39 Screening of different types of sugars or polyhydroxy alcohols, antioxidant protection measures, and surfactant dosages

[0156] The stability of each formulation was investigated at different temperatures (40 / 25 / 5℃), and sample solutions were taken at different time points for testing. The purity of the active ingredient antibody (size exclusion chromatography SEC purity, capillary isoelectric focusing icIEF purity) and hyaluronidase activity were evaluated to investigate the chemical and physical stability of the antibody and the hyaluronidase activity.

[0157] Table 40 Purity Results at 40℃

[0158] Table 41 Purity Results at 25℃

[0159] Table 42 Purity Results at 5℃

[0160] Table 43 Results of Surfactant Content

[0161] Tables 40-43 show that using different amounts of poloxamer 188 has no significant effect on protein purity. Formulations using poloxamer 188 and polysorbate 20 surfactant have no significant effect on protein purity. However, in stability studies, the poloxamer 188 content remained essentially unchanged in the formulations using poloxamer 188, while the polysorbate 20 content decreased to varying degrees in stability studies at different temperatures, potentially leading to risks to formulation stability and safety.

[0162] Each formulation sample was subjected to a freeze-thaw cycle test (placed at -20℃ for 2 days, then heated to 5℃ and placed for 2 days, which is considered one cycle), and the cycle was repeated 5 times to investigate stability.

[0163] Table 44 Purity Results under Freeze-Thaw Conditions

[0164] Table 44 shows that sucrose, trehalose, and sorbitol all have the same protein protection effect during freezing.

[0165] Table 45 Results of Hyaluronidase Activity

[0166] Table 45 shows that the hyaluronidase activity of prescriptions 2 and 6 remained unchanged after accelerated storage at 25°C and long-term storage at 5°C for 6 months.

[0167] Based on the above data, the final selected drug composition contains 120 mg / mL daratumumab; 2000 U / mL hyaluronidase; 8 mM histidine buffer with a pH range of 5.5; 250 mM trehalose; 0.02% w / v to 0.06% (w / v) surfactant poloxamer 188 and 0.9 mg / mL stabilizer methionine. This formulation exhibited the best stability in the comprehensive test.

Claims

1. A pharmaceutical composition suitable for subcutaneous injection, comprising: CD38 monoclonal antibodies ranging from 10 mg / mL to 180 mg / mL; Hyaluronidase ranging from 50 U / ml to 5000 U / ml; 1 mM to 50 mM histidine buffer, pH range 5.0-7.0; 50 mM to 400 mM of polyhydroxy alcohols or sugars, selected from sorbitol, trehalose or sucrose; 0.01% (w / v) to 2% (w / v) of the surfactant poloxamer 188; and Methionine, a stabilizer, at concentrations ranging from 0.1 mg / mL to 2.5 mg / mL.

2. The pharmaceutical composition according to claim 1, characterized in that: The CD38 monoclonal antibody in the pharmaceutical composition is selected from daratumumab, isatuximab, TJ202, HexaBody-CD38, SAR-442085 or CID-103.

3. The pharmaceutical composition according to claim 1, characterized in that: The CD38 monoclonal antibody mentioned is daratum.

4. The pharmaceutical composition according to claim 3, characterized in that: The host protein content in the daratumumab is <1 ng / mg.

5. The pharmaceutical composition according to claim 1, characterized in that: In the pharmaceutical composition, the concentration of CD38 monoclonal antibody is 50 mg / ml-150 mg / ml, 100 mg / ml-150 mg / ml, 100 mg / ml-130 mg / ml, or 120 mg / ml.

6. The pharmaceutical composition according to claim 1, characterized in that: In the pharmaceutical composition described above, hyaluronidase can be derived from animal or human samples or manufactured using recombinant DNA technology.

7. The pharmaceutical composition according to claim 6, characterized in that: The hyaluronidase mentioned is animal-derived Dessau, Vitrase™, Hydase™, Amphadase™, recombinant human hyaluronidase rHuPH20, or its mutants.

8. The pharmaceutical composition according to claim 1, characterized in that: The amount of hyaluronidase in the pharmaceutical composition is 100 U / ml to 4000 U / ml, 500 U / ml to 3000 U / ml, 1500 U / ml to 2500 U / ml, or 2000 U / ml.

9. The pharmaceutical composition according to claim 1, characterized in that: The pharmaceutical composition contains 5 mM to 30 mM buffered histidine with a pH range of 5.0-7.0, 5 mM to 20 mM buffered histidine with a pH range of 5.0-7.0, 5 mM to 10 mM buffered histidine with a pH range of 5.0-6.0, or 8 mM buffered histidine with a pH of 5.

5.

10. The pharmaceutical composition according to claim 1, characterized in that: The pharmaceutical composition contains 100 mM to 300 mM of a polyhydroxy alcohol or sugar selected from sorbitol, trehalose or sucrose, 150 mM to 300 mM of trehalose or sucrose, 200 mM to 300 mM of trehalose or sucrose, or 250 mM of trehalose dihydrate.

11. The pharmaceutical composition according to claim 1, characterized in that: The pharmaceutical composition contains 0.01% (w / v) to 1% (w / v) of the surfactant poloxamer 188, 0.01% (w / v) to 0.1% (w / v) of the surfactant poloxamer 188, or 0.02% (w / v) to 0.06% (w / v) of the surfactant poloxamer 188.

12. The pharmaceutical composition according to claim 1, characterized in that: The pharmaceutical composition comprises 0.5 mg / mL to 2.5 mg / mL of stabilizer methionine, 0.5 mg / mL to 2 mg / mL of stabilizer methionine, 0.5 mg / mL to 1 mg / mL of stabilizer methionine, or 0.9 mg / mL of stabilizer methionine.

13. A pharmaceutical composition suitable for subcutaneous injection, comprising: Daratumumab at concentrations ranging from 10 mg / mL to 180 mg / mL; Hyaluronidase ranging from 50 U / ml to 5000 U / ml; 1 mM to 50 mM histidine buffer, pH range 5.0-7.0; 50 mM to 400 mM of polyhydroxy alcohols or sugars, selected from sorbitol, trehalose or sucrose; 0.01% (w / v) to 2% (w / v) of the surfactant poloxamer 188; and Methionine, a stabilizer, at concentrations ranging from 0.1 mg / mL to 2.5 mg / mL.

14. A pharmaceutical composition suitable for subcutaneous injection, comprising: daratumumab at concentrations ranging from 50 mg / mL to 150 mg / mL; Hyaluronidase at concentrations ranging from 500 U / ml to 3000 U / ml; 5mM to 30mM histidine buffer, pH range 5.0-7.0; 50 mM to 400 mM of polyhydroxy alcohols or sugars, selected from sorbitol, trehalose or sucrose; 0.01% (w / v) to 1% (w / v) of the surfactant poloxamer 188; and Methionine, a stabilizer, at concentrations ranging from 0.5 mg / mL to 2 mg / mL.

15. A pharmaceutical composition suitable for subcutaneous injection, comprising: daratumumab at concentrations of 100 mg / mL to 150 mg / mL; Hyaluronidase at concentrations ranging from 500 U / ml to 3000 U / ml; 5 mM to 20 mM histidine buffer, pH range 5.0-7.0; 100mM to 300mM of polyhydroxy alcohols or sugars, selected from sorbitol, trehalose or sucrose; 0.01% (w / v) to 1% (w / v) of the surfactant poloxamer 188; and Methionine, a stabilizer, at concentrations ranging from 0.5 mg / mL to 1.5 mg / mL.

16. A pharmaceutical composition suitable for subcutaneous injection, comprising: daratumumab at concentrations ranging from 100 mg / mL to 130 mg / mL; Hyaluronidase at concentrations ranging from 1500 U / ml to 2500 U / ml; 5mM to 10mM histidine buffer, pH range 5.0-6.0; 100mM to 300mM of trehalose or sucrose; 0.01% (w / v) to 1% (w / v) of the surfactant poloxamer 188; and Methionine, a stabilizer, at concentrations ranging from 0.5 mg / mL to 1.0 mg / mL.

17. A pharmaceutical composition suitable for subcutaneous injection, comprising: 120 mg / mL daratumumab; 2000 U / ml hyaluronidase; 8 mM histidine buffer, pH 5.5; 250mM trehalose dihydrate; Surfactant poloxamer 188 from 0.02% w / v to 0.06% (w / v); and Methionine, a stabilizer, at a concentration of 0.9 mg / mL.

18. The pharmaceutical composition according to any one of claims 1-17, characterized in that: The dosage volume of the drug composition containing daratum is 5-20 ml, 12-18 ml, or 15 ml.

19. Use of the pharmaceutical composition according to any one of claims 1-17 in the preparation of a medicament for treating a disease, wherein the CD38 monoclonal antibody pharmaceutical composition may induce the killing of CD38-expressing tumor cells by antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), apoptosis, or in vitro regulation of CD38 enzyme activity.

20. Use of the pharmaceutical composition according to any one of claims 1-17 in the preparation of a medicament for treating a disease, wherein the CD38 monoclonal antibody pharmaceutical composition can mediate antitumor efficacy through an immunomodulatory effect, said immunomodulatory effect being mediated by inducing CD4+. + and CD8 + Cell proliferation, and / or the suppression of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs)-mediated inflammatory responses.

21. The use according to claim 19 or 20, wherein the disease is multiple myeloma (MM), smoldering multiple myeloma (SMM), glioblastoma, ITP, plasma cell myeloma, lupus erythematosus, lupus nephritis, Waldenstrom macroglobulinemia, prostate cancer, membranous proliferative glomerulonephritis, malignant tumors of primary lymphoid hematopoiesis, RR-T cell lymphoma, hemolytic anemia, relapsed or refractory chronic lymphocytic leukemia, plasmablastic lymphoma, non-small cell lung cancer, or Alzheimer's disease.

22. The use according to claim 19 or 20, characterized in that: The pharmaceutical composition is administered in combination with a second therapeutic agent or a combination thereof.

23. The use according to claim 22, characterized in that: The second therapeutic agent is an alkylating agent, which may be selected from busulfan, cyclophosphamide, bendamustine, levofloxacin, carboplatin, cisplatin, temozolomide, melphalan, carmustine, cyclohexanenitrosourea, dacarbazine, oxaliplatin, ifosfamide, dichloroethylmethylamine, thiotepa, trabectedin, or streptozotocin.

24. The use according to claim 22, characterized in that: The second therapeutic agent is a proteasome inhibitor selected from bortezomib, carfilzomib, or ixazomib.

25. The use according to claim 22, characterized in that: The second therapeutic agent is a glutamate derivative selected from lenalidomide, thalidomide, or pomalidomide.

26. The use according to claim 22, characterized in that: The second therapeutic agent is a corticosteroid selected from dexamethasone or prednisone.

27. The use according to any one of claims 22-26, characterized in that: The pharmaceutical composition may be administered simultaneously or sequentially with a second therapeutic agent or in combination thereof.

28. The use according to any one of claims 19-26, characterized in that: In the pharmaceutical composition, the CD38 monoclonal antibody is daratumumab.

29. The use according to claim 28, characterized in that: The daratumumab drug composition described herein can be used in combination with bortezomib, lenalidomide, and dexamethasone for induction and consolidation therapy in newly diagnosed patients suitable for autologous stem cell transplantation.

30. The use according to claim 28, characterized in that: The daratumumab drug composition described herein can be used in combination with lenalidomide and dexamethasone (Rd) to treat newly diagnosed MM patients who are not suitable for autologous hematopoietic stem cell transplantation, as well as relapsed or refractory MM patients who have received at least one first-line treatment.

31. The use according to claim 28, characterized in that: The daratumumab drug composition described herein can be used in combination with bortezomib and dexamethasone for the treatment of patients with multiple myeloma who have received at least one prior therapy.

32. The use according to claim 28, characterized in that: The daratumumab drug composition described herein can be used in combination with bortezomib, melphalan, and prednisone to treat newly diagnosed MM patients who are not suitable for autologous hematopoietic stem cell transplantation.

33. The use according to claim 28, characterized in that: The daratumumab drug composition described herein can be used in combination with carfilzomib and dexamethasone to treat patients with relapsed or refractory multiple myeloma who have previously received one to three treatments.

34. The use according to claim 28, characterized in that: The daratumumab drug composition described herein can be used in combination with pomalidomide and dexamethasone for the treatment of patients with multiple myeloma who have received at least one first-line therapy, including lenalidomide and proteasome inhibitor therapy.

35. The use according to claim 28, characterized in that: The daratumumab drug composition described herein can be used in combination with bortezomib, thalidomide, and dexamethasone for the treatment of newly diagnosed patients with multiple myeloma who are eligible for autologous hepatocyte transplantation.

36. The use according to claim 28, characterized in that: The daratumumab drug composition can be used in combination with bortezomib, cyclophosphamide, and dexamethasone to treat newly diagnosed patients with primary light chain amyloidosis.

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