Highly concentrated antibody compositions

Abstract

Methods for producing an aqueous antibody composition comprising at least 200 mg / ml antibody and methods for producing a dried antibody composition are provided. High concentration antibody compositions are further provided. Further provided is the use of the provided antibody composition in medicine.

Description

Technical Field

[0001] Provided are methods for producing aqueous antibody compositions comprising at least 200 mg / ml of antibody and methods for producing dried antibody compositions. Further provided are high concentration antibody compositions. Further provided are uses of the provided antibody compositions in medicine. Background Art

[0002] Antibodies are multifunctional components of the immune system. In the past few decades, a large number of monoclonal antibodies have been approved as pharmaceutical products. Monoclonal antibodies (or fragments thereof) are produced against different antigens and used in medicine, for example, for the treatment of immune diseases and cancer.

[0003] The marginal stability of antibodies (especially highly concentrated antibodies), combined with the expectation of long-term stability, has always been the driving force for the development of methods that can stabilize proteins. Dehydration of protein formulations to produce dry powders and the addition of excipients are the two most common stabilization techniques for extending shelf life. Protein stability can be significantly enhanced by removing water from formulations. This is the result of reduced protein mobility and the absence of certain degradation pathways promoted by water. Under suitable excipients and drying conditions, proteins are incorporated into an amorphous matrix, and as long as the temperature remains below the glass transition temperature, this amorphous matrix can be maintained, thereby enhancing the long-term stability of proteins. In addition, cryoprotectants (such as trehalose and sucrose) stabilize proteins by excluding water near the protein, and provide protection against degradation during the drying process, when the dry powder is stored, and when the aqueous solution is stored after rehydration. In order to avoid denaturation at the air-water interface, surfactants are usually used to prevent the adsorption of proteins at the interface.

[0004] Antibody formulations with good long-term stability and appropriate powder properties are needed. The long-term stability of dry powders depends primarily on the water content (typically less than 5%), formulation, protein structure, and storage conditions. Although drying is generally necessary to obtain a long-term stable product, the drying process may introduce a combination of thermal, interfacial, and mechanical stresses that may negatively affect protein stability. Optimized drying process parameters and the addition of excipients are needed to reduce the impact of these stresses on the product. The addition of excipients changes the effect of the drying process and makes the process protein and formulation specific.

[0005] Several techniques can be used to dry proteins and obtain protein powders. This includes drying techniques such as freeze drying or lyophilization, spray drying, spray freeze drying or granulation and supercritical fluid drying. All of these techniques use different physical principles for drying, induce different stresses during the drying process, and also produce powders with different particle or powder properties.

[0006] Gikanga et al. disclosed a method for producing high-concentration monoclonal antibody formulations via spray drying (Gikanga et al. PDA J Pharm Sci Technol. 2015 Jan-Feb;69(1):59-73).

[0007] Deokar et al. provided a comparison of different techniques for obtaining high-concentration (approximately 200 mg / mL) antibody formulations (Deokar et al. J Pharm Sci. 2020; 109(12): 3579-3589).

[0008] Clénet D, Hourquet V, Woinet B, Ponceblanc H, Vangelisti MA spray freezedried micropellet based formulation proof-of-concept for a yellow fever vaccine candidate. Eur J Pharm Biopharm. 2019 Sep;142:334-343.

[0009] WO 2018 / 204374 A1 discloses aqueous antibody formulations.

[0010] US2020 / 390705 A1 discloses spray-dried antibody formulations.

[0011] US 11 351 256 B2, WO 2013 / 016648 A2 and WO 2013 / 063510 A1 disclose methods for obtaining antibody formulations.

[0012] However, high concentrations of pharmaceutical antibody formulations may present many issues, such as stability challenges (e.g., formation of visible and subvisible particles and antibody aggregates) and solution property challenges (e.g., high turbidity). It is important that antibody formulations for parenteral use have low aggregate levels (up to 5% high molecular weight species or acceptable HMWS according to generally accepted industry norms), low subvisible particles (in accordance with USP <787> Specifications, allowable levels: 10μm particles ≤ 6000 particles / container, 25μm particles ≤ 600 particles / container), low turbidity (up to 40 NTU based on marketed product benchmarks, Kingsbury JS et al. J Pharm Sci. 2021 Sep;110(9):3176-3182. doi:10.1016 / j.xphs.2021.05.005. Epub 2021 May 15. PMID:34004217.) and no visible particles. USP <787> It is a product of the United States Pharmacopeial (USP) convention USP <787> Chapter 1, Standards for Subvisible Particulate Matter in Therapeutic Protein Injections.

[0013] Therefore, it is important to avoid the formation of antibody aggregates (which can be measured by measuring the change in the percentage of high molecular weight species). While it is important that the final antibody formulation used by the end user has stability attributes that meet specifications, the stability of the antibody during drying, during storage of dry intermediate products (e.g., powders or pellets) at the recommended storage temperature and shelf life, and during storage as a solution at the recommended storage temperature and shelf life must be ensured in order to have a commercially viable product.

[0014] Highly concentrated antibody drug formulations, such as formulations containing at least 200 mg / ml of antibody, are needed, particularly if large doses of the antibody are to be administered, such as by subcutaneous administration using a prefilled syringe. The ability to obtain formulations containing 200 mg / mL or more of the antibody would allow for higher amounts of antibody drug substance in a single prefilled syringe, and thus higher doses to be administered via subcutaneous administration rather than intravenous administration, or in some cases to reduce the number of subcutaneous injections, both of which would enhance patient-centric therapy.

[0015] Therefore, there remains a need for improved high concentration drug antibody compositions that avoid these limitations and meet typical acceptability specifications. Summary of the invention

[0016] Provided herein is a method for producing an aqueous antibody composition I comprising at least 200 mg / ml antibody, the method comprising steps a), b) and c)

[0017] a) providing an aqueous composition A comprising

[0018] a1) an antibody, the concentration of which is lower than the concentration of the antibody in the antibody composition I,

[0019] a2) at least one surfactant,

[0020] a3) at least one cryoprotectant,

[0021] a4) at least 0.4% (w / v) arginine or a salt thereof, wherein the weight (w) is calculated as arginine based on the total volume of the aqueous composition A, for example 0.4% to 3.5% (w / v) arginine, wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8, and optionally

[0022] a5) Buffer

[0023] b) drying the aqueous composition A provided in step a) to produce an antibody composition B, and

[0024] c) reconstitute the composition dried in step b) to give the aqueous antibody composition I.

[0025] In embodiments of the provided methods, the salt of arginine is selected from the group consisting of arginine-Cl, arginine-aspartate, arginine-glutamate, arginine-sulfate, arginine-acetate, arginine-succinate, and mixtures thereof.

[0026] In embodiments of the provided methods, the arginine is arginine-Cl.

[0027] In embodiments of provided methods, at least one cryoprotectant is selected from the group consisting of trehalose, sucrose, sorbitol, glycerol, and mannitol.

[0028] In embodiments of the provided methods, the cryoprotectant is trehalose.

[0029] In embodiments of the provided methods, the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:4, such as 11:2 to 11:4.

[0030] In embodiments of provided methods, aqueous composition A comprises less than 200 mg / ml antibody, e.g., 10-190 mg / ml antibody, e.g., 20-170 mg / ml antibody, e.g., 50-150 mg / ml antibody, e.g., 80-120 mg / ml antibody.

[0031] In examples of the provided methods, aqueous composition A comprises 150 mg / ml antibody.

[0032] In embodiments of the provided methods, aqueous antibody composition I comprises greater than 200 mg / ml antibody.

[0033] In embodiments of the provided methods, aqueous antibody composition I comprises at least 250 mg / ml antibody, such as more than 250 mg / ml antibody.

[0034] In embodiments of the provided methods, aqueous antibody composition I comprises at least 300 mg / ml antibody, such as more than 300 mg / ml antibody.

[0035] In embodiments of the provided methods, aqueous antibody composition I comprises 230 mg / ml to 420 mg / ml antibody, e.g., 260 mg / ml to 400 mg / ml antibody.

[0036] In embodiments of the provided methods, aqueous antibody composition I comprises 270 to 350 mg / ml antibody, e.g., 280 to 320 mg / ml antibody, e.g., 300 mg / ml antibody.

[0037] In embodiments of the provided methods, the antibody is a monoclonal antibody.

[0038] In embodiments of provided methods, at least one surfactant is a polysorbate, such as polysorbate 20 or 80, or a poloxamer, such as poloxamer 188.

[0039] In embodiments of the provided methods, the buffer is a histidine, citrate, acetate, phosphate, Tris, succinate, or glycine buffer.

[0040] In embodiments of the provided methods, the pH of Composition A is about 5.0 to 8.0, such as about 5.5 to 7.

[0041] In embodiments of the provided methods, the antibody composition B is in the form of a powder or pellets.

[0042] In embodiments of the provided methods, step b) is performed by freeze drying or spray drying to give a freeze dried or spray dried powder.

[0043] In embodiments of the provided methods, step b) is performed by spray freeze drying to give spray freeze dried micropellets.

[0044] In embodiments of the provided methods, the freeze-dried powder is reconstituted in the reconstitution solution for a period of 1.5 to 4.0 hours.

[0045] In embodiments of the provided methods, the spray-dried powder is reconstituted in a reconstitution solution for a period of at least four hours.

[0046] In embodiments of the provided methods, the spray freeze-dried micropellets are reconstituted in a reconstitution solution over a period of 1 to 2 hours.

[0047] In some embodiments, the powder or pellets are reconstituted in water. Thus, the reconstitution solution can be water.

[0048] In embodiments of the provided methods, the aqueous antibody composition I is a clear and transparent solution.

[0049] Further provided is an aqueous antibody composition I obtained or obtainable by a method for producing an aqueous antibody composition provided herein.

[0050] Further provided is an aqueous antibody composition I comprising at least 200 mg / ml antibody.

[0051] In the provided examples of aqueous antibody compositions I, the aqueous antibody composition I comprises 270 to 350 mg / ml antibody, such as 280 to 320 mg / ml antibody, such as 300 mg / ml antibody.

[0052] In another embodiment of the provided aqueous antibody composition I, the aqueous antibody composition I comprises 230 mg / ml to 420 mg / ml antibody, for example 260 mg / ml to 400 mg / ml antibody.

[0053] In embodiments, the aqueous antibody composition I is a clear and transparent solution.

[0054] In an embodiment, the aqueous antibody composition I comprises at least 200 mg / mL antibody and has an opalescence of less than 40 NTU, such as less than 25 NTU or less than 15 NTU.

[0055] In an embodiment, the aqueous antibody composition I further comprises at least one surfactant, at least one cryoprotectant (eg, trehalose), and arginine or a salt thereof.

[0056] Further provided herein is a container comprising an aqueous antibody composition I provided herein.

[0057] In the embodiment of the container provided, the container is a pre-filled syringe, a vial, a cartridge, an ampoule or an auto-injector. For example, the container can be an auto-injector or a pre-filled syringe.

[0058] Also provided herein are kits comprising a container provided herein, and a label or instructions for administering and using the aqueous antibody composition I.

[0059] Further provided is an aqueous antibody composition I provided herein for use in medicine.

[0060] Also provided is a method for producing a dried antibody composition B,

[0061] a) providing an aqueous composition A comprising

[0062] a1) less than 200 mg / ml antibody, such as 10 to 190 mg / ml antibody, such as 20 to 170 mg / ml antibody, such as 50 to 150 mg / ml antibody, such as 80 to 120 mg / ml antibody,

[0063] a2) at least one surfactant,

[0064] a3) at least one cryoprotectant, and

[0065] a4) at least 0.4% (w / v) arginine or a salt thereof, wherein the weight (w) is calculated as arginine based on the total volume of the aqueous composition A, e.g. 0.4% to 3.5% (w / v) arginine,

[0066] wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8, and

[0067] b) spray freeze-drying the composition provided in step a) to give the dried antibody composition B.

[0068] In an embodiment of the above method, the dried antibody composition B is in the form of micropellets.

[0069] Also provided are a dried antibody composition B obtained or obtainable by a method for producing a dried antibody composition B and a container comprising the dried antibody composition B.

[0070] Also provided is a dried antibody composition B and a container, the container comprising the dried antibody composition B, the dried antibody composition B is in the form of micropellets, and the composition B comprises

[0071] a1) Antibodies,

[0072] a2) at least one surfactant,

[0073] a3) at least one cryoprotectant, such as trehalose, and

[0074] a4) arginine or a salt thereof,

[0075] wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8, such as 11:2 to 11:8, wherein the weight of a4 is calculated as free amino acid.

[0076] Also provided is a dried antibody composition B and a container, the container comprising the dried antibody composition B, the dried antibody composition B is in the form of a powder, and the composition B comprises

[0077] a1) Antibodies,

[0078] a2) at least one surfactant,

[0079] a3) at least one cryoprotectant, such as trehalose, and

[0080] a4) arginine or a salt thereof,

[0081] wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8, such as 11:2 to 11:8, wherein the weight of a4 is calculated as free amino acid.

[0082] Further provided is the use of a dried antibody composition B provided herein for producing an aqueous antibody composition I as defined herein comprising at least 200 mg / ml antibody.

[0083] Further provided is the use of an aqueous composition A for the production of an aqueous antibody composition I as defined herein comprising at least 200 mg / ml antibody.

[0084] Further provided is an aqueous composition A as defined in connection with a method for producing an aqueous antibody composition I. The definitions apply accordingly.

[0085] Therefore, an aqueous composition A is provided, comprising:

[0086] a1) less than 200 mg / ml antibody, such as 10 to 190 mg / ml antibody, such as 20 to 170 mg / ml antibody, such as 50 to 150 mg / ml antibody, such as 80 to 120 mg / ml antibody, a2) at least one surfactant,

[0087] a3) at least one cryoprotectant, and

[0088] a4) at least 0.4% (w / v) arginine or a salt thereof, wherein the weight (w) is calculated as arginine based on the total volume of the aqueous composition A, for example 0.4% to 3.5% (w / v) arginine, wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] Figure 1 Reconstituted solutions of eight different freeze-dried monoclonal antibodies at concentrations of 100, 150, 200, 250, 300, 350 and 400 mg / mL. Clear and transparent solutions up to 300 mg / mL were obtained for all eight antibodies.

[0090] Figure 2 Percentage of high molecular weight species (a) and opalescence (b) of 100-350 mg / mL reconstituted solutions of eight mAbs. Marginal changes in high molecular weight species were observed with increasing mAb concentration, with all values ​​below 5%. Decreases in opalescence were observed with increasing mAb concentration, with all values ​​<30 NTU.

[0091] Figure 3 Increase in the percentage of high molecular weight species during freeze drying. %HMWS was measured before and after freeze drying. A marginal increase in %HMWS was observed due to freeze drying, indicating a protective effect of the excipient. Compositions containing at least 0.5% Arginine-Cl generally had a lower increase in HMWS relative to compositions containing only trehalose.

[0092] Figure 4 Increase in percentage of high molecular weight species of freeze-dried powders after storage at 40°C. Compositions containing at least 0.5% arginine-Cl generally had a lower increase in HMWS relative to compositions containing trehalose alone. Although 40°C is not an expected storage condition, it is generally considered a condition for accelerated stability testing.

[0093] Figure 5 Reconstitution solutions with 300 mg / mL monoclonal antibodies A, B, D and E. An overview of the formulations can be found in Table 2. All antibodies and compositions shown resulted in clear and transparent aqueous solutions with a target protein concentration of 300 mg / mL.

[0094] Figure 6 Change in % HMWS between Antibody Composition I (300 mg / mL) and Antibody Composition A (100 mg / mL) for monoclonal antibodies A, B, D, and E. Figure 2 Similar to the observations presented in , marginal changes in high molecular weight species were observed with increasing mAb concentrations.

[0095] Figure 7 Changes in % HMWS between antibody compositions I (300 mg / mL) after 2 weeks of storage at 40° C. Marginal changes in high molecular weight species were observed after short-term storage at 40° C. Although 40° C. is not an expected storage condition, it is generally considered a condition for accelerated stability testing.

[0096] Figure 8Reconstitution solutions with 300 mg / mL monoclonal antibodies A, B, D, E, G and H after reconstitution using various aqueous solutions. An overview of the formulations can be found in Example 3. All antibodies and compositions shown resulted in clear and transparent aqueous solutions with a target protein concentration of 300 mg / mL, indicating that various aqueous solutions are suitable for reconstitution.

[0097] Fig. 9 Changes in % HMWS of antibody composition I (300 mg / mL) after two weeks of storage at 40°C. An overview of the formulation can be found in Example 3. Reconstituted solutions containing glycine, arginine-Cl, arginine-glutamate, magnesium-glutamate, a combination of arginine-Cl and glycine, and a combination of arginine-Cl and magnesium-glutamate were effective in providing protection. Marginal changes in high molecular weight species were observed after short-term storage at 40°C. 40°C is not an expected storage condition and is generally considered a condition for accelerated stability testing.

[0098] Fig.10 Spray freeze-dried micropellets and spray dried powders for different formulations of monoclonal antibodies I and B. An overview of the formulations can be found in Example 4.

[0099] Fig.11 Composition I (300 mg / mL) after reconstitution of spray-freeze-dried micropellets and spray-dried powders of different formulations for monoclonal antibodies I and B. After reconstitution of spray-freeze-dried micropellets with opalescence <5 NTU of antibody B, clear and transparent aqueous solutions with a target antibody concentration of 300 mg / mL were obtained for both antibodies. After reconstitution of the spray-dried powders, 300 mg / mL antibody solutions were obtained for both antibodies, and the resulting solutions were turbid and had opalescence >30 NTU.

[0100] Fig.12 The spray-dried powder and spray-freeze-dried micropellets were evaluated for short-term and long-term storage stability at different temperatures. Marginal changes in high molecular weight species were observed, with all values ​​below 5%, indicating optimal storage stability.

[0101] Fig.13 Evaluation of short-term storage and accelerated stability of 300 mg / mL Composition I produced by spray-dried powder reconstitution and spray freeze-dried at different temperatures and for different storage periods. Marginal changes in high molecular weight species were observed at 5°C and 25°C, with all values ​​below 5%, indicating optimal storage stability. The high starting value for Antibody B can be attributed to analytical variability and sample handling.

[0102] definition

[0103] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. It is noteworthy that, as used in this specification and the appended claims, the singular forms "a / an" and "the" also include plural referents unless the context clearly indicates otherwise. In addition, it should be understood that, as used herein, the term "at least one" means one or more than one. For example, two, three, four or more. Depending on the item to which the term refers, the technician understands the upper limit (if any) that the term can refer to.

[0104] As used herein, the term "about" means that there is an interval of precision about any number listed after the term, within which the technical effect can be achieved. Therefore, as mentioned herein, "about" refers to, for example, an exact value or a range of ±20%, ±15%, ±10% or ±5% of the exact value. In an embodiment, the term refers to an exact value.

[0105] As used herein, the term "comprising" should not be understood in a limiting sense. The term is rather intended to indicate that there may be more than the actual items mentioned, e.g., if it refers to a method comprising certain steps, the presence of additional steps should not be excluded. However, the term "comprising" also encompasses embodiments in which only the mentioned items are present, i.e., it has a limiting meaning in the sense of "consisting of."

[0106] The aqueous composition A provided in step a) of the method of the present invention should comprise antibodies.

[0107] As used herein, the term "antibody" refers to an immunoglobulin or immunoglobulin-like molecule. Antibodies include, for example, but are not limited to, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate (e.g., mammals such as goats, rabbits, and mice, and non-mammalian species such as shark immunoglobulins). The term includes synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, single domain antibodies (also known as immunoglobulin single variable domains (ISVDs), e.g., Molecules) (including monospecific, bispecific, multispecific single domain antibodies), chimeric antibodies, intracellular antibodies, single chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), camelized antibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies, and epitope binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., antigen binding domains or molecules containing antigen binding sites that specifically bind to antigens (e.g., the six complementary determining regions (CDRs) of antibodies). Antibodies can belong to any class of immunoglobulin molecules (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b). In some embodiments, the antibody is humanized. In certain embodiments, the antibody is an IgG antibody, such as an IgG1 or IgG4 antibody. In embodiments, the antibody is an IgG4 antibody.

[0108] In embodiments, the antibody or fragment thereof has been recombinantly produced.

[0109] The light chain consists of two domains or regions: a variable domain (VL) and a constant domain (CL). The heavy chain consists of four domains: a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine the binding recognition and specificity for the antigen. The light chain constant region domain (CL) and the heavy chain constant region domain (CH) confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of the immunoglobulin Fab fragment and consists of the variable portion of one light chain and one heavy chain. The specificity of an antibody lies in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site consists of residues primarily from the hypervariable region or the complementarity determining region (CDR). Sometimes, residues from non-hypervariable regions or framework regions (FR) affect the overall domain structure and thus the binding site. The term "complementarity determining region" (abbreviated as CDR) refers to the amino acid sequence that together determines the binding affinity and specificity of the native Fv region of a natural immunoglobulin binding site. The light chain and heavy chain of an immunoglobulin each have three CDRs, designated as CDR1-L, CDR2-L, CDR3-L (for the light chain complementary determining region) or CDRL1, CDRL2, CDRL3 and CDR1-H, CDR2-H, CDR3-H (for the heavy chain complementary determining region) or CDRH1, CDRH2, CDRH3. Therefore, a conventional antibody antigen-binding site includes six CDRs, which include sets of CDRs from each heavy chain variable region and light chain variable region.

[0110] In an embodiment, the antibody is a monoclonal antibody. As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of a B lymphocyte or an antibody produced by a cell transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those skilled in the art, such as by preparing hybrid antibody-forming cells by a fusion of myeloma cells and immune spleen cells.

[0111] In an embodiment, the antigen binding protein is a diabody. A diabody is a bivalent antibody comprising two polypeptide chains. Each polypeptide chain comprises a variable heavy chain domain and a variable light chain domain connected by a joint. In an embodiment, the two polypeptide chains of the diabody are identical. In an alternative embodiment, the two polypeptide chains have different amino acid sequences, provided that the two chains bind to the same antigen (the same epitope or different epitopes within the same antigen).

[0112] The term "antibody" may also encompass mixtures of antibodies, such as a mixture of two or more antibodies.

[0113] In an embodiment, the antibody is a therapeutic antibody, i.e., an antibody used in therapy. For example, the antibody is selected from the group consisting of adalimumab, belimumab, atezolizumab, elotuzumab, infliximab, evolocumab, nivolumab, cetuximab, pembrolizumab, durvalumab, siltuximab, eculizumab, remanezumab, ofatumumab, omalizumab, caneluzumab, rituximab, panitumumab, daratumumab, parituzumab, Lipizumab, denosumab, mepolizumab, golimumab, trastuzumab, obinutuzumab, avelumab, omeclizumab, pertuzumab, vedolizumab, ramucirumab, tocilizumab, secukinumab, olaratumumab, necituzumab, guselkumab, ustekinumab, bevacizumab, alemtuzumab, certolizumab pegol (pecilizumab), and benralizumab.

[0114] In addition to the antibody, the "composition" or "formulation" should optionally contain additional ingredients in the amounts specified herein. Excipients refer to inert substances commonly used as diluents, vehicles, preservatives, binders, stabilizers, etc. for drugs, and include, but are not limited to, amino acids (e.g., arginine), fatty acids and phospholipids, surfactants (e.g., polysorbates, nonionic surfactants, etc.), sugars (e.g., sucrose, maltose, trehalose, etc.). "Pharmaceutically acceptable excipients" means any inert substance that is combined with the antibodies mentioned herein to prepare an acceptable or convenient dosage form. "Pharmaceutically acceptable excipients" are excipients that are non-toxic to the recipient at the dosages and concentrations used and are compatible with the other ingredients of the formulation (including monoclonal antibodies).

[0115] In addition to a1) the antibody, composition A further comprises a2) at least one surfactant, a3) at least one cryoprotectant, a4) arginine or a salt thereof. In addition, the composition may comprise a5) a buffer. Typically, the composition comprises an excipient in an amount as specified elsewhere herein.

[0116] Surfactants are chemical compounds that interact with biomolecules and / or general pharmaceutical excipients in formulations and stabilize them. Surfactants generally protect adhesives from stress induced by air / solution interface and stress induced by solution / surface, otherwise these stresses may cause protein aggregation. Surfactants may include but are not limited to polysorbates, glycerol, dicarboxylic acids, oxalic acid, succinic acid, fumaric acid, phthalic acid and combinations thereof. It is known to those skilled in the art that other surfactants, such as nonionic or ionic detergents, may be used as long as they are pharmaceutically acceptable, i.e., suitable for application to a subject. In certain embodiments, the surfactant is a polysorbate. Examples of polysorbates include polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80. In an embodiment, the polysorbate is polysorbate 80. In an embodiment, the polysorbate is polysorbate 20. Polysorbate 80, polysorbate 20, and poloxamer 188 are the most commonly used surfactants in commercially available parenteral products.

[0117] In some embodiments, the surfactant is a poloxamer, such as poloxamer 188.

[0118] Typically, the surfactant is present in composition A in an amount of about 0.001% to about 0.2% (w / v), based on the total volume of composition A. For example, the surfactant may be present in composition A in an amount of about 0.005% (w / v), about 0.006% (w / v), about 0.007% (w / v), about 0.008% (w / v), about 0.009% (w / v), about 0.01% (w / v), about 0.02% (w / v), about 0.03% (w / v), about 0.04% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v), about 0.09% (w / v), about 0.1% (w / v), and about 0.2% (w / v). In some embodiments, the surfactant is present in formulation A at about 0.01% to about 0.1% (w / v), about 0.02% to about 0.08% (w / v), or about 0.03% to about 0.07% (w / v), such as about 0.05% (w / v). For example, polysorbate 80 can be present in an amount of about 0.05% (w / v).

[0119] Since the surfactant is present in composition A, it will also be present in composition I.

[0120] Cryoprotectants are chemical compounds that protect the antibodies contained in the composition from freezing damage (i.e., damage caused by ice formation or adsorption to the ice-liquid interface). Thus, cryoprotectants provide stability to the antibodies in the formulation to counteract freezing-induced stresses. Cryoprotectants can be sugars (e.g., trehalose, sucrose, glucose, mannitol, mannose, and lactose) or polymers (e.g., or polyethylene glycol). Typically, cryoprotectants are selected from the group consisting of cryoprotectants consisting of trehalose, sucrose, sorbitol, glycerol, and mannitol.

[0121] In some embodiments, the cryoprotectant is trehalose.

[0122] The formulation or aqueous composition mentioned herein should contain the amino acid arginine or its salt. Typically, the composition contains a salt of arginine. In an embodiment, the arginine salt is selected from the group consisting of arginine-Cl (also referred to as arginine-HCl), arginine-aspartate, arginine-glutamate, arginine-sulfate, arginine-acetate, arginine-succinate and mixtures thereof.

[0123] In embodiments, the salt is arginine-Cl.

[0124] The aqueous composition may contain a5) a buffer. A buffer is an agent that maintains a physiologically suitable pH. In addition, the buffer may enhance the isotonicity and chemical stability of the formulation. For example, the buffer may be selected from histidine, citrate, acetate, phosphate, Tris, succinate or glycine buffer.

[0125] In some embodiments, the buffer is present in a concentration of about 0.5 mM to about 50 mM (e.g., about 5 mM to about 15 mM) in composition A. For example, the buffer can be present at a concentration of about 15 mM.

[0126] In certain embodiments, the composition A provided in step a) of the method provided herein can have a pH ranging from about 5.0 to 8.0 (e.g., about 5.5 to about 7.0). For example, the pH of composition A can be about 5.5, about 5.7, about 5.9, about 6.1, about 6.1, about 6.3, about 6.5, about 6.7, about 6.9, and about 7.0. In certain embodiments, the pH of composition A can range from about 5.8 to about 6.3. The pH of composition A can be measured by any method known to those skilled in the art. The method for measuring pH is to use a pH meter. The pH of composition A can be adjusted using any method known in the art. Chemicals for changing the pH of composition A are hydrochloric acid (HCl) and sodium hydroxide (NaOH).

[0127] In some embodiments, the pH of composition A is antibody specific, i.e., selected to provide optimal stability. For example, the pH may be lower or higher than the isoelectric point (pi) of the antibody. The isoelectric point is the pH at which a particular molecule or surface does not carry a net charge. In an embodiment, the pH of composition A is not at the isoelectric point of the antibody. For example, the pH of composition A may be at least 0.5 higher or lower than the isoelectric point of the antibody.

[0128] In step b) of the method of the present invention, the composition A provided in step a) should be dried to produce the antibody composition B.

[0129] As used herein, the term "drying" or "dehydration" refers to the removal of water from an aqueous composition A, thereby generating a composition B. Typically, the water content (and therefore the moisture content) of composition B is less than 10%, such as less than 5%, such as less than 3%. In an embodiment, the water content is between 0.5% and 5%, such as between 0.5% and 3%. The water content or moisture content is the amount of water contained in composition B, typically measured by Karl-Fischer moisture analysis. Lower moisture content can be achieved by carefully selecting the drying process parameters. Lower moisture content will ensure better stability during storage of composition B. In the studies on which the present invention is based, Karl Fischer was used to determine the residual water content. The oven temperature was set to 120°C and the air flow was set to 80mL / min, and the drift needed to be less than 20μg water / minute to start the measurement. The stop criterion was a relative drift close to 15μg water / minute. Hydranal coulomat AG-oven (Honeywell Fluka) reagent was used as the anolyte in the titration cell. Before testing the samples, a 1% water standard (MilliporeSigma) was run to ensure that the instrument was functioning properly. In the examples, the moisture content mentioned herein was measured by this method.

[0130] Drying can be achieved by any method deemed suitable, for example, it can be achieved by techniques such as freeze drying (or lyophilization), spray drying and spray freeze drying. Typically, drying is achieved by freeze drying, spray drying or spray freeze drying.

[0131] In an embodiment, the drying step is performed by freeze drying (also often referred to as "lyophilization").

[0132] As used herein, the term "freeze-drying" (or "lyophilization" or "cryodesiccation") refers to a method in which composition A is cooled to a temperature at which the water in the composition is frozen. The frozen water is then removed by one or two drying steps (a primary drying step (which involves removing unbound water by sublimation) and optionally a subsequent secondary drying step (which involves removing bound solvent by desorption)).

[0133] In another embodiment, the drying step is performed by spray drying.

[0134] Spray drying is a three-step process involving an atomization step, followed by removal of the aqueous solvent by evaporation and subsequent powder collection. The protein feed solution is sprayed directly into a drying chamber where the droplets are contacted with a hot drying gas (such as air or nitrogen). The drying time is very short and can vary from a few seconds to a few minutes depending on the scale of the spray dryer. After drying, the powder is collected by a cyclone separator or filter. Process parameters (such as feed flow rate, drying air flow rate, atomization flow rate and inlet temperature) and formulation parameters (such as excipients and solid concentration) affect water content, particle size, particle morphology, density and protein stability. Mechanical stress during atomization, thermal stress during drying and interfacial stress during overall processing may have an adverse effect on product stability. An increase in drying temperature will reduce the moisture content, which will help protein stability, but will also introduce more thermal stress and potential denaturation.

[0135] In another embodiment, the drying step is performed by spray freeze drying.

[0136] Spray freeze drying (often also referred to as "granulation") is a four-step process involving an atomization step, spray freezing of droplets to "lock" the spherical droplet shape, then freeze drying and subsequent collection of micropellets (see, e.g., WO 2013 / 050156 A1 or WO 2009 / 109550 A1). Although the physical and morphological properties of the resulting powder are primarily affected by the first two steps, the third step involves the drying time and total energy required for the dried product. Process parameters (e.g., feed flow rate, nozzle configuration, freezing rate, freezing temperature, drying air flow rate and pressure) and formulation parameters (e.g., excipients and solid concentration) affect water content, particle size, particle morphology, density and protein stability. Mechanical stress during atomization, ice-related stress during freezing, thermal stress during drying and interfacial stress during overall processing may have an adverse effect on product stability. This technology allows a high degree of control of residual moisture content, mass density and particle size. Examples of spray freeze drying are disclosed in the Examples section.

[0137] The composition B produced in step b) can be a powder (i.e., a dried powder) or in the form of micropellets. For example, the micropellets can be regular spherical micropellets or particles with a diameter of about 400 μm to about 700 μm. The diameter can be measured by scanning electron microscopy (SEM).

[0138] The form of composition B may depend on the drying method. In an embodiment, step b) is carried out by freeze drying or spray drying to give a (freeze dried or spray dried) powder. In another embodiment, step b) is carried out by spray freeze drying to give spray freeze dried micropellets.

[0139] In an example, composition B is reconstituted to give an aqueous antibody composition I.

[0140] In step c) of the method of the invention, the dried composition B is reconstituted to give an aqueous antibody composition I, ie a composition comprising at least 200 mg / ml antibody.

[0141] Step c) may be performed immediately after composition B has been obtained. In another embodiment, composition B is stored for a period of time before step c) is performed.

[0142] Reconstitution of composition B may be carried out in any aqueous (reconstitution) solution deemed suitable (eg, an aqueous buffer).

[0143] In embodiments, the reconstitution solution is an aqueous solution comprising at least one additive selected from the group consisting of water, glycine, arginine-Cl, guanidine-Cl, arginine-glutamate, and magnesium-glutamate.

[0144] Aqueous antibody composition I or composition B mentioned herein should be a "stable" composition. A stable composition is a composition in which the antibody substantially maintains its physical stability, characteristics, integrity and / or chemical stability, characteristics, integrity and / or biological activity during storage. Various analytical techniques for measuring protein stability are available in the art. Stability can be measured for a selected time period under selected temperature and other storage conditions. Stability can be determined by characterizing the aggregation or level of high molecular weight substances using size exclusion chromatography. For example, if there is no sign of a significant increase in aggregation level, precipitation and / or denaturation when visually inspecting color and / or clarity and characterizing the level of high molecular weight substances, the antibody "maintains its physical stability" in the composition. In certain embodiments, when using the composition of the present invention, 5% or less, typically 4% or less, typically 3% or less, more typically 2% or less, and particularly 1% or less of the antibody forms aggregates, as measured by size exclusion chromatography (e.g., as described in the example section) or any other suitable method for measuring aggregate formation. For example, if the antibody monomer has a purity of about 90% or more, typically about 95% or more, or about 98% or more after a certain predetermined period of time in a specific formulation under certain storage conditions, the antibody is considered to be stable in a specific composition. In an embodiment, less than 5% of the antibody forms aggregates. Chemical stability can be assessed by detecting and quantitatively modifying the chemical form of the protein. Chemical changes can involve size changes (e.g., shearing), which can be assessed using, for example, (HP) SEC, SDS-PAGE and / or matrix-assisted laser desorption ionization / time of flight mass spectrometry (MALDI / TOF MS). Other types of chemical changes include charge changes that can be assessed by, for example, ion exchange chromatography.

[0145] The term "opalescence" (often also referred to as "turbidity") refers to an optical characteristic of a solution caused by light scattering, which can be explained by different scattering events (e.g., Rayleigh scattering or Mie scattering). The opalescence of a solution can be evaluated, for example, as described in Kingsbury JS et al. J Pharm Sci. [Journal of Pharmaceutical Sciences] 2021 Sep; 110(9):3176-3182.doi:10.1016 / j.xphs.2021.05.005. Epub 2021 May 15. PMID:34004217 (incorporated by reference in its entirety). In an embodiment, the opalescence of composition A and aqueous antibody composition I mentioned herein is less than 40 NTU (scattering turbidity units (NTU)), for example, the opalescence is less than 25 NTU. For example, the opalescence can be less than 15 NTU. In an embodiment, the opalescence of the dried antibody composition A mentioned herein is less than 40 NTU (nephelometric turbidity units (NTU)), for example, the opalescence is less than 25 NTU. For example, the opalescence may be less than 15 NTU. In an embodiment, the opalescence of the aqueous antibody composition I mentioned herein is less than 40 NTU (nephelometric turbidity units (NTU)), for example, the opalescence is less than 25 NTU. For example, the opalescence may be less than 15 NTU.

[0146] The aqueous antibody composition I or composition B mentioned can be used in therapy. Thus, the composition can be administered to a subject in need thereof, i.e. a subject suffering from a disease or disorder or at risk of suffering from a disease or disorder.

[0147] If composition B is to be used in therapy, the composition needs to be reconstituted to produce an aqueous antibody composition I as described elsewhere herein.

[0148] In one embodiment, the term "disease or disorder" refers to any pathological or unhealthy state that can be treated by administering a formulation or composition provided herein.

[0149] A "subject" or "patient" can be a vertebrate. The term "subject" includes humans and other animals, particularly mammals and other organisms. Thus, herein, a subject can be an animal, such as a mouse, rat, hamster, rabbit, guinea pig, ferret, cat, dog, chicken, sheep, bovine species, horse, camel, or primate. In some embodiments, the subject is a mammal. In some embodiments, the subject is a primate. In some embodiments, the subject is a human.

[0150] The term "administer" or "administration" refers to the act of injecting or otherwise physically delivering a substance (e.g., a formulation of the present invention) present outside the body to a patient, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. In an embodiment, the composition is administered subcutaneously. In another embodiment, the composition is administered intravenously.

[0151] The compositions described herein are generally administered in a therapeutically effective amount. The term "therapeutically effective amount" is understood by the skilled artisan. In some embodiments, the term refers to an amount that achieves a desired therapeutic response or desired therapeutic effect alone or in combination with another dose, optionally without causing or only minimally causing unacceptable or unwanted side effects.

[0152] As used herein, the term "treating" or "treatment" refers to administering a compound or composition or a combination of compounds or compositions to a subject to: prevent, improve or eliminate a disease and / or disorder mentioned herein, such as obesity, of the subject. Therefore, the term encompasses the treatment of an existing disease or disorder mentioned herein, or the prevention, i.e., prophylaxis, of a disease or disorder. Therefore, it will be appreciated that in some embodiments, the treatment mentioned herein may be preventive. In some embodiments, the term refers to the treatment of an existing disease or disorder mentioned herein. Therefore, the subject suffers from the disease or disorder. DETAILED DESCRIPTION

[0153] Provided herein is a method for producing an aqueous antibody composition I comprising at least 200 mg / ml antibody, the method comprising steps a), b) and c),

[0154] a) providing an aqueous composition A comprising

[0155] a1) an antibody, the concentration of which is lower than the concentration of the antibody in the antibody composition I,

[0156] a2) at least one surfactant,

[0157] a3) at least one cryoprotectant,

[0158] a4) at least 0.4% (w / v) arginine or a salt thereof, wherein the weight (w) is calculated as arginine, e.g. 0.4% to 3.5% (w / v) arginine,

[0159] wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8, and

[0160] b) drying the composition provided in step a) to produce antibody composition B, and

[0161] c) reconstitution of the composition dried in step b),

[0162] To give the aqueous antibody composition I.

[0163] The aqueous antibody composition I produced by the method of the present invention should contain a high concentration of antibodies, such as monoclonal antibodies, typically therapeutic monoclonal antibodies.

[0164] Typically, the aqueous antibody composition I comprises at least 200 mg / ml antibody, such as more than 200 mg / ml antibody.

[0165] Also typically, the aqueous antibody composition I comprises at least 250 mg / ml antibody, such as at least 300 mg / ml antibody.

[0166] In an embodiment, the aqueous antibody composition I comprises 230 mg / ml to 420 mg / ml antibody, for example 260 mg / ml to 400 mg / ml antibody.

[0167] In an embodiment, the aqueous antibody composition I comprises 270 to 350 mg / ml antibody, for example 280 to 320 mg / ml antibody.

[0168] In an embodiment, aqueous antibody composition I comprises 300 mg / ml antibody.

[0169] Typically, the aqueous antibody composition I is a clear and transparent solution. This can be assessed visually (by the appearance of the composition).

[0170] The provided method for producing an aqueous antibody composition I should include the following steps:

[0171] a) Providing an aqueous composition A

[0172] b) drying the composition provided in step a) to produce antibody composition B, and

[0173] c) reconstitution of the composition dried in step b),

[0174] To give the aqueous antibody composition I.

[0175] The aqueous composition provided in step a) should comprise:

[0176] a1) an antibody, the concentration of which is lower than the concentration of the antibody in the antibody composition I,

[0177] a2) at least one surfactant,

[0178] a3) at least one cryoprotectant,

[0179] a4) at least 0.4% (w / v) arginine or a salt thereof, wherein the weight (w) is calculated as arginine, e.g. 0.4% to 3.5% (w / v) arginine,

[0180] wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is from 11:1 to 11:8, and optionally

[0181] a5) Buffer.

[0182] The concentration of the antibody in a1) should be lower than the concentration of the antibody in the aqueous antibody composition I. For example, if the concentration of the antibody in composition I is at least 200 mg / ml, the concentration of the antibody in composition A is lower than 200 mg / ml. In some embodiments, the concentration of the antibody in composition A is at least 5%, such as at least 10%, such as at least 20%, such as at least 20%, lower than the concentration of the antibody in composition I. For example, the concentration of the antibody in composition A is 10% to 70%, such as 30% to 60% lower than the concentration in composition I.

[0183] In an embodiment, aqueous composition A comprises less than 200 mg / ml antibody.

[0184] In an embodiment, aqueous composition A comprises 10 to 190 mg / ml antibody.

[0185] In an embodiment, aqueous composition A comprises 20 to 170 mg / ml antibody.

[0186] In an embodiment, aqueous composition A comprises 50 to 150 mg / ml antibody.

[0187] In an embodiment, aqueous composition A comprises 80 to 120 mg / ml antibody.

[0188] In an embodiment, aqueous composition A comprises 100 mg / ml antibody.

[0189] The term "surfactant" has been defined above. In addition, typical concentrations of surfactants are given above. In an embodiment, at least one surfactant is a polysorbate, such as polysorbate 80. In another embodiment, the surfactant is a poloxamer, such as poloxamer 188.

[0190] The term "cryoprotectant" has been defined above. For example, the cryoprotectant is selected from the group of cryoprotectants consisting of trehalose, sucrose, sorbitol, glycerol and mannitol. In an embodiment, the cryoprotectant is trehalose.

[0191] The aqueous composition A may comprise a5) a buffer as described above.

[0192] In embodiments, the buffer is a histidine buffer.

[0193] In an alternative embodiment, the buffer is a citrate buffer.

[0194] In an alternative embodiment, the buffer is acetate buffer.

[0195] In an alternative embodiment, the buffer is phosphate buffer.

[0196] In an alternative embodiment, the buffer is Tris buffer.

[0197] In an alternative embodiment, the buffer is a succinate buffer.

[0198] In an alternative embodiment, the buffer is a glycine buffer.

[0199] Furthermore, in embodiments, the pH of the composition is about 5.0 to 8.0, such as about 5.5 to 7 (but may depend on the antibody).

[0200] Typically, the aqueous composition A comprises a4) at least 0.4% (w / v) arginine or a salt thereof, calculated as arginine based on the total volume of the composition A. For example, the aqueous composition A comprises 0.4% to 3.5% (w / v) arginine (as free base). In an embodiment, the aqueous composition A comprises 1.0% to 3.0% (w / w) arginine (as free base), for example 1.25% to 2.8% (w / w) arginine.

[0201] It will be appreciated that where the aqueous composition A comprises an arginine salt, the amount is also calculated as the amount of arginine free base present. The molecular weight of arginine free base is 174 g / mol. The term "amount" typically refers to weight.

[0202] In an embodiment, the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8. For example, the ratio may be about 11:1, about 11:2, about 11:3, about 11:4, about 11:5, about 11:6, about 11:7, about 11:8. The ratio of the amount of a1) (i.e., the amount of the antibody) to the sum of the amounts of a3) and a4) (i.e., the amount of the excipient) should be understood as a weight / weight (w / w) ratio.

[0203] In an embodiment, the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:4.

[0204] In an embodiment, the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:2 to 11:4.

[0205] In an embodiment, the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:2 to 11:3.

[0206] In an embodiment, the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:4.

[0207] In some embodiments, a4) is a salt of arginine. In one embodiment, the presence of at least 0.4% arginine in composition A was shown to enhance the stability of the antibody during the drying process (see Examples).

[0208] In embodiments, the arginine salt is arginine-Cl (arginine-HCl, see also Examples section).

[0209] In alternative embodiments, the arginine salt is arginine-aspartate.

[0210] In alternative embodiments, the arginine salt is arginine-glutamate.

[0211] In alternative embodiments, the arginine salt is arginine-sulfate.

[0212] In alternative embodiments, the arginine salt is arginine-acetate.

[0213] In alternative embodiments, the arginine salt is arginine-succinate.

[0214] In step b) of the method of the invention, aqueous composition A produces antibody composition B. After drying, the water content of the dried composition (ie composition B) is less than 10%, such as less than 5%, such as less than 3%.

[0215] In an embodiment, composition A is dried by freeze drying. Freeze drying typically produces a powder. Therefore, composition B is provided in the form of a powder.

[0216] In an alternative embodiment, composition A is dried by spray drying. Spray drying typically produces a powder. Therefore, composition B is provided in the form of a powder.

[0217] In an alternative embodiment, composition A is dried by spray freeze drying. Spray freeze drying typically produces a powder. Therefore, composition B is provided in the form of micropellets.

[0218] In step c) of the method of the invention, the dried composition B is reconstituted to give an aqueous antibody composition I, ie a composition comprising at least 200 mg / ml antibody.

[0219] Step c) can be carried out immediately after composition B has been obtained. However, it is also envisaged to store composition B for a period of time, since the studies on which the present invention is based have shown that composition B has long-term stability (see Examples). For example, composition B can be stored for at least one month, for example at least three months, for example at least six months. In an embodiment, the composition is stored for one to three months. In an alternative embodiment, composition B is stored for three to twelve months, for example three to six months. Composition B can be stored at a temperature between 2°C and 25°C, for example at a temperature of 5°C to 25°C. Typically, composition B is stored refrigerated at a temperature of 2°C to 8°C, or stored at a temperature of between 17°C and 23°C under ambient conditions.

[0220] Reconstitution of Composition B can be carried out in any aqueous (reconstitution) solution deemed suitable (e.g., aqueous buffer). In the studies described in the Examples section, Composition B was successfully reconstituted in the following aqueous reconstitution solutions:

[0221] 1.5% or 200 mM glycine,

[0222] 3% or 150 mM Arginine-Cl,

[0223] 6.4% or 200 mM arginine-glutamate,

[0224] 5.8% or 150 mM magnesium-glutamate,

[0225] 3% or 150 mM arginine-Cl and 1.5% or 200 mM glycine, and

[0226] 3% or 150 mM arginine-Cl and 5.8% or 150 mM magnesium-glutamate.

[0227] In an embodiment, the reconstitution solution is water, i.e., sterile water. Thus, the powder or pellets are reconstituted in water.

[0228] To produce composition B, a volume of aqueous reconstitution buffer is used which achieves the desired antibody concentration of the antibody in composition I, ie a volume which produces composition I having the desired antibody concentration (eg a concentration of 300 mg / ml).

[0229] It should be understood that the reconstitution of composition B in aqueous reconstitution requires a certain reconstitution time. In an embodiment, the reconstitution is carried out for a period of time sufficient to dissolve the antibody in the aqueous solution. Advantageously, it has been shown that the reconstitution time of the spray-freeze-dried composition mentioned herein is shorter than the reconstitution time of the spray-dried or freeze-dried composition B.

[0230] In the examples of the provided methods, the dried antibody composition B has been obtained by spray drying and is thus a spray-dried powder. Typically, the spray-dried powder is reconstituted in a reconstitution solution for a period of at least four hours.

[0231] In the examples of the provided methods, the dried antibody composition B has been obtained by freeze drying and is thus a freeze-dried powder. Typically, the freeze-dried powder is reconstituted in a reconstitution solution for a period of 1.5 to 4.0 hours.

[0232] In the examples of the provided methods, the dried antibody composition B has been obtained by spray freeze drying and is therefore in the form of spray freeze dried micropellets. Typically, the spray freeze dried micropellets are reconstituted in a reconstitution solution for a period of 1 to 2 hours.

[0233] The short reconstitution time allows the final composition I to be prepared shortly before the composition is administered. For example, the composition I can be prepared by medical personnel, such as in a hospital. The short reconstitution time also allows the preparation of a highly concentrated aqueous solution that is directly filled into a vial or a prefilled syringe. For example, the composition I can be prepared by reconstitution of the composition B and filled into a vial or a prefilled syringe in a manufacturing plant and sold as a ready-to-use liquid product.

[0234] However, the present invention is not limited to preparing composition I shortly before application. Since composition I is stable, it can be prepared in advance, for example by a manufacturer of a dry composition. Subsequently, composition I can be stored for an extended period of time. For example, composition B can be stored under refrigerated or ambient storage conditions for at least one month, such as at least three months, such as at least six months. In an embodiment, the composition is stored for one to three months. In an alternative embodiment, composition I is stored for three to twelve months, such as three to six months. Typically, composition B is stored refrigerated at a temperature between 2°C and 8°C.

[0235] The definitions and explanations given above apply mutatis mutandis to the following.

[0236] Further provided is a container comprising the aqueous antibody composition I provided herein.

[0237] In an embodiment, the container is a pre-filled syringe.

[0238] In an alternative embodiment, the container is a vial.

[0239] In an alternative embodiment, the container is an autoinjector.

[0240] The container can include any volume of aqueous antibody composition I deemed suitable. In an embodiment, the container includes about 1 to 4 ml of composition I, such as 1 to 2 ml of composition I. For example, the container can include about 1 ml, 2 ml, 3 ml or 4 ml of composition I. The volume can depend on the type of container. For example, a vial can include a volume of up to 50 ml, wherein a prefilled syringe can include a volume of up to 2 ml.

[0241] Also provided are kits comprising a container as provided herein, and, optionally, a label or instructions for administering and using the aqueous antibody composition I or the dried antibody composition B.

[0242] If the container comprises a dried antibody composition B, the kit may further comprise a reconstitution buffer. Typically, the kit comprises a volume of reconstitution buffer sufficient to produce composition I.

[0243] Further provided is the use of the aqueous antibody composition I or the dried antibody composition B provided herein for use in medicine.

[0244] Further provided is a method for producing a dried antibody composition B,

[0245] a) providing an aqueous composition A comprising

[0246] a1) antibody, as shown in conjunction with the method for producing composition I, the antibody concentration is based on the total volume of composition A, for example less than 200 mg / ml antibody, such as 10 to 190 mg / ml antibody, such as 20 to 170 mg / ml antibody, such as 50 to 150 mg / ml antibody, such as 80 to 120 mg / ml antibody.

[0247] a2) at least one surfactant,

[0248] a3) at least one cryoprotectant, and

[0249] a4) at least 0.4% (w / v) arginine or a salt thereof, wherein the weight (w) is calculated as arginine, e.g. 0.4% to 3.5% (w / v) arginine,

[0250] wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8, and

[0251] b) spray freeze-drying the composition provided in step a),

[0252] To give the dried antibody composition B.

[0253] In embodiments, the dried antibody composition is in the form of micropellets.

[0254] A dried antibody composition B obtained or obtainable by a method for producing a dried antibody composition B is further provided.

[0255] Further provided is a dried antibody composition B in the form of micropellets, the composition B comprising a1) an antibody (as defined herein),

[0256] a2) at least one surfactant (as defined herein),

[0257] a3) at least one cryoprotectant (as defined herein), for example trehalose, and a4) arginine or a salt thereof (as defined herein),

[0258] wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8, such as 11:2 to 11:8, wherein the weight of a4 is calculated as free amino acid.

[0259] In some embodiments, the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:4, such as 11:2 to 11:4.

[0260] Further provided is a dry antibody composition B in the form of a powder, the composition B comprising a1) an antibody (as defined herein),

[0261] a2) at least one surfactant (as defined herein),

[0262] a3) at least one cryoprotectant (as defined herein), for example trehalose, and a4) arginine or a salt thereof (as defined herein),

[0263] wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8, such as 11:2 to 11:8, wherein the weight of a4 is calculated as free amino acid.

[0264] In some embodiments, the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:4, such as 11:2 to 11:4.

[0265] Further provided is a container comprising a dried antibody composition B provided herein. For example, the container can be a vial, a prefilled syringe, a cartridge, an ampoule or an automatic injector. In some embodiments, the container is a prefilled syringe or an automatic injector.

[0266] Also provided is the use of the dried antibody composition B provided herein for producing an aqueous antibody composition I comprising at least 200 mg / ml antibody (eg, more than 200 mg / ml antibody).

[0267] Further provided is an aqueous antibody composition I as described herein. The composition can be used in medicine.

[0268] In an embodiment, the aqueous antibody composition I comprises 270 to 350 mg / ml antibody, for example 280 to 320 mg / ml antibody.

[0269] In further embodiments, the aqueous antibody composition I comprises at least 250 mg / ml antibody, such as at least 300 mg / ml antibody.

[0270] In another embodiment of the provided aqueous antibody composition I, the aqueous antibody composition I comprises 230 mg / ml to 420 mg / ml antibody, for example 260 mg / ml to 400 mg / ml antibody.

[0271] In an embodiment, aqueous antibody composition I comprises about 300 mg / ml antibody.

[0272] Typically, the aqueous antibody composition I has an opalescence of less than 40 NTU (nephelometric turbidity units (NTU)), for example an opalescence of less than 25 NTU. For example, the opalescence may be less than 15 NTU.

[0273] In an embodiment, the aqueous antibody composition I further comprises at least one surfactant, at least one cryoprotectant (eg, trehalose), and arginine or a salt thereof.

[0274] In some embodiments, the aqueous antibody composition I is obtained or obtainable by a method for producing an aqueous antibody composition I provided herein.

[0275] Additionally, there is provided an aqueous composition A as defined in conjunction with a method for producing an aqueous antibody composition I comprising at least 200 mg / ml antibody, said composition A comprising

[0276] a1) less than 200 mg / ml antibody, such as 10 to 190 mg / ml antibody, such as 20 to 170 mg / ml antibody, such as 50 to 150 mg / ml antibody, such as 80 to 120 mg / ml antibody, a2) at least one surfactant,

[0277] a3) at least one cryoprotectant, and

[0278] a4) at least 0.4% (w / v) arginine or a salt thereof, wherein the weight (w) is calculated as arginine based on the total volume of the aqueous composition A, for example 0.4% to 3.5% (w / v) arginine, wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8.

[0279] The definitions and explanations given for composition A in conjunction with the method for producing an aqueous antibody composition apply mutatis mutandis.

[0280] Example

[0281] An overview of the provided embodiments is provided below. The definitions and explanations given above apply mutatis mutandis to the following.

[0282] 1. A method for producing an aqueous antibody composition I comprising at least 200 mg / ml antibody, wherein the method comprises the following steps:

[0283] a) providing an aqueous composition A comprising

[0284] a1) an antibody, the concentration of which is lower than the concentration of the antibody in the antibody composition I,

[0285] a2) at least one surfactant,

[0286] a3) at least one cryoprotectant,

[0287] a4) at least 0.4% (w / v) arginine or a salt thereof, wherein the weight (w) is calculated as arginine, e.g. 0.4% to 3.5% (w / v) arginine,

[0288] wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8, and

[0289] b) drying the composition provided in step a) to produce antibody composition B, and

[0290] c) reconstitution of the composition dried in step b),

[0291] To give the aqueous antibody composition I.

[0292] 2. The method of embodiment 1, wherein the salt of arginine is arginine-Cl, arginine-aspartate, arginine-glutamate, arginine-sulfate, arginine-acetate, arginine-succinate.

[0293] 3. The method of embodiment 2, wherein the salt of arginine is arginine-Cl.

[0294] 4. The method of any one of embodiments 1 to 3, wherein the at least one cryoprotectant is selected from the group of cryoprotectants consisting of trehalose, sucrose, sorbitol, glycerol, and mannitol.

[0295] 5. The method of embodiment 4, wherein the cryoprotectant is trehalose.

[0296] 6. The method of any one of embodiments 1 to 5, wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:4, such as 11:2 to 11:4.

[0297] 7. The method of any one of embodiments 1 to 6, wherein the aqueous composition A comprises less than 200 mg / ml antibody, such as 10 to 190 mg / ml antibody, such as 20 to 170 mg / ml antibody, such as 50 to 150 mg / ml antibody, such as 80 to 120 mg / ml antibody.

[0298] 8. The method of any one of embodiments 1 to 7, wherein composition I comprises more than 200 mg / ml antibody, e.g., 230 mg / ml to 420 mg / ml antibody, e.g., 260 mg / ml to 400 mg / ml antibody.

[0299] 9. The method of embodiment 8, wherein composition I comprises 270 to 350 mg / ml antibody, e.g. 280 to 320 mg / ml antibody, e.g. 300 mg / ml antibody.

[0300] 10. The method of any one of embodiments 1 to 9, wherein the antibody is a monoclonal antibody.

[0301] 11. The method of any one of embodiments 1 to 10, wherein the at least one surfactant is a polysorbate, such as polysorbate 20 or 80, or a poloxamer, such as poloxamer 188.

[0302] 12. The method of any one of embodiments 1 to 11, wherein composition A further comprises a5) a buffer, such as a histidine, citrate, acetate, phosphate, Tris, succinate, or glycine buffer.

[0303] 13. The method of any one of embodiments 1 to 12, wherein the pH of composition A is about 5.0 to 8.0, such as about 5.5 to 7.

[0304] 14. The method of any one of embodiments 1 to 13, wherein the antibody composition B is in the form of a powder or pellets.

[0305] 15. The method of any one of embodiments 1 to 14, wherein step b) is performed by freeze drying or spray drying to give a freeze dried or spray dried powder.

[0306] 16. The method of embodiment 15, wherein the freeze-dried powder is reconstituted in the reconstitution solution for a period of 1.5 to 4.0 hours, or wherein the spray-dried powder is reconstituted in the reconstitution solution for a period of at least four hours.

[0307] 17. The method of any one of embodiments 1 to 14, wherein step b) is performed by spray freeze drying to give spray freeze dried micropellets.

[0308] 18. The method of embodiment 17, wherein the spray freeze-dried micropellets are reconstituted in a reconstitution solution for a period of 1 to 2 hours.

[0309] 19. The method of any one of embodiments 1 to 18, wherein the aqueous antibody composition I is a clear and transparent solution.

[0310] 20. An aqueous antibody composition I obtained or obtainable by the method of any one of embodiments 1 to 19.

[0311] 21. A container comprising the aqueous antibody composition I as described in Example 20.

[0312] 22. The container of embodiment 21, wherein the container is a prefilled syringe, vial, cartridge, ampoule, or autoinjector.

[0313] 23. A kit comprising the container of embodiment 21 or 22, and a label or instructions for administering and using the aqueous antibody composition I.

[0314] 24. The aqueous antibody composition I of embodiment 21, for use in medicine.

[0315] 25. A method for producing a dried antibody composition B,

[0316] a) providing an aqueous composition A comprising

[0317] a1) less than 200 mg / ml antibody, such as 10 to 190 mg / ml antibody, such as 20 to 170 mg / ml antibody, such as 50 to 150 mg / ml antibody, such as 80 to 120 mg / ml antibody,

[0318] a2) at least one surfactant,

[0319] a3) at least one cryoprotectant, and

[0320] a4) at least 0.4% (w / v) arginine or a salt thereof, wherein the weight (w) is calculated as arginine, e.g. 0.4% to 3.5% (w / v) arginine,

[0321] wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8, and

[0322] b) spray freeze-drying the composition provided in step a),

[0323] To give the dried antibody composition B.

[0324] 26. The method of embodiment 25, wherein the dried antibody composition is in the form of micropellets.

[0325] 27. A dried antibody composition B obtained or obtainable by the method described in Example 25 or 26.

[0326] 28. A container comprising the dried antibody composition B as described in Example 27.

[0327] 29. Use of the dried antibody composition as described in Example 26 or 27 for producing an aqueous antibody composition I comprising at least 200 mg / ml antibody.

[0328] 30. An aqueous antibody composition I comprising at least 200 mg / ml antibody.

[0329] 31. The aqueous antibody composition I of embodiment 30, comprising 270 to 350 mg / ml antibody, for example 280 to 320 mg / ml antibody, for example 300 mg / ml antibody.

[0330] 32. The aqueous antibody composition I of embodiment 30, comprising 230 mg / ml to 420 mg / ml antibody, for example 260 mg / ml to 400 mg / ml antibody.

[0331] 33. The aqueous antibody composition I of any one of embodiments 30 to 32, wherein the composition is a clear and transparent solution.

[0332] 34. The aqueous antibody composition I of any one of embodiments 30 to 33, having an opalescence of less than 40 NTU (nephelometric turbidity units (NTU)), such as an opalescence of less than 25 NTU or less than 15 NTU.

[0333] 35. The aqueous antibody composition I of any one of embodiments 30 to 34, comprising at least one surfactant, at least one cryoprotectant, and arginine or a salt thereof.

[0334] 36. The aqueous antibody composition I of embodiment 35, wherein the at least one cryoprotectant is trehalose.

[0335] 37. The aqueous antibody composition I of embodiment 35 or 36, wherein the at least one surfactant is a polysorbate, such as polysorbate 20 or 80, or a poloxamer, such as poloxamer 188.

[0336] 38. The aqueous antibody composition I of any one of embodiments 30 to 37, comprising histidine.

[0337] 39. The aqueous antibody composition I of any one of embodiments 30 to 38, further comprising glycine and / or magnesium-glutamate.

[0338] 40. A dried antibody composition B in the form of micropellets, the composition B comprising

[0339] a1) an antibody (as defined herein),

[0340] a2) at least one surfactant (as defined herein),

[0341] a3) at least one cryoprotectant (as defined herein), such as trehalose, and

[0342] a4) arginine or a salt thereof (as defined herein),

[0343] wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8, such as 11:2 to 11:8, wherein the weight of a4 is calculated as free amino acid.

[0344] 41. A dry antibody composition B in powder form, the composition B comprising

[0345] a1) an antibody (as defined herein),

[0346] a2) at least one surfactant (as defined herein),

[0347] a3) at least one cryoprotectant (as defined herein), such as trehalose, and

[0348] a4) arginine or a salt thereof (as defined herein),

[0349] wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8, such as 11:2 to 11:8, wherein the weight of a4 is calculated as free amino acid.

[0350] 42. Use of the dried antibody composition as described in Example 40 or 41 for producing an aqueous antibody composition I comprising at least 200 mg / ml antibody.

[0351] 43. An aqueous composition A, comprising

[0352] a1) less than 200 mg / ml antibody, such as 10 to 190 mg / ml antibody, such as 20 to 170 mg / ml antibody, such as 50 to 150 mg / ml antibody, such as 80 to 120 mg / ml antibody,

[0353] a2) at least one surfactant,

[0354] a3) at least one cryoprotectant, and

[0355] a4) at least 0.4% (w / v) arginine or a salt thereof, wherein the weight (w) is calculated as arginine based on the total volume of the aqueous composition A, for example 0.4% to 3.5% (w / v) arginine, wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8.

[0356] 44. The aqueous composition A as described in embodiment 43, wherein the salt of arginine is arginine-Cl, arginine-aspartate, arginine-glutamate, arginine-sulfate, arginine-acetate, arginine-succinate.

[0357] 45. The aqueous composition A as described in embodiment 44, wherein the salt of arginine is arginine-Cl.

[0358] 46. ​​The aqueous composition A of any one of embodiments 43 to 45, wherein the at least one cryoprotectant is selected from the group of cryoprotectants consisting of trehalose, sucrose, sorbitol, glycerol and mannitol.

[0359] 47. The aqueous composition A of embodiment 46, wherein the cryoprotectant is trehalose.

[0360] 48. The aqueous composition A of any one of embodiments 43 to 47, wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:4, such as 11:2 to 11:4.

[0361] 49. An aqueous composition A as described in any one of embodiments 43 to 48, wherein the aqueous composition A comprises less than 200 mg / ml antibody, such as 10 to 190 mg / ml antibody, such as 20 to 170 mg / ml antibody, such as 50 to 150 mg / ml antibody, such as 80 to 120 mg / ml antibody.

[0362] 50. The aqueous composition A of any one of embodiments 43 to 49, wherein the antibody is a monoclonal antibody.

[0363] 51. The aqueous composition A of any one of embodiments 43 to 50, wherein the at least one surfactant is a polysorbate, such as polysorbate 20 or 80, or a poloxamer, such as poloxamer 188.

[0364] 52. The aqueous composition A of any one of embodiments 43 to 51, wherein composition A further comprises a5) a buffer, such as a histidine, citrate, acetate, phosphate, Tris, succinate or glycine buffer.

[0365] 53. The aqueous composition A of any one of embodiments 43 to 52, wherein the pH value of the composition A is about 5.0 to 8.0, such as about 5.5 to 7.

[0366] 54. Use of the aqueous antibody composition A as described in any one of claims 43 to 53 for producing an aqueous antibody composition I comprising at least 200 mg / ml antibody.

[0367] All references cited throughout the specification are incorporated herein for the disclosure specifically mentioned above and in their entirety.

[0368] The invention will now be further described by reference to the following examples, which are intended to illustrate but not to limit the scope of the invention.

[0369] Examples

[0370] Materials and methods

[0371] Physical stability assessment by size exclusion chromatography (SEC)

[0372] Physical stability is assessed by regularly measuring the amount of aggregates generated in the monoclonal antibody formulation. Samples are measured by eluting about 100 μg of total monoclonal antibody from a TSK-Gel G3000SWXL analytical column (Tosoh Co.) with 40 mM phosphate, 150 mM sodium chloride (pH 7.2) (detection wavelength: 280 nm). Aggregates are designated as high molecular weight substances (HMWS) that elute faster than non-aggregated mAb monomers; HMWS% is calculated by dividing the amount of aggregates generated by the sum of total aggregated monoclonal antibody molecules + non-aggregated monoclonal antibody molecules.

[0373] SoloVPE for protein concentration

[0374] Total protein concentration was determined by measuring the absorbance at 280 nm using a Lunatic instrument (Unchained Labs). Samples of the powder group were measured without any dilution, and samples of the liquid group were measured after dilution 10 to 15 times in formulation buffer. Two measurements were performed for each 3 μL sample, and the average value was reported. The antibody-specific molar extinction coefficient was used for concentration calculation. Results were considered comparable when the difference was equal to or less than 10%.

[0375] Opalescence

[0376] The samples were analyzed according to the protocol established in Kingsbury JS et al. J Pharm Sci. 2021 Sep; 110(9): 3176-3182. doi: 10.1016 / j.xphs.2021.05.005. Epub 2021 May 15. PMID: 34004217. Briefly, the samples were analyzed by μ-turbidimetry using the “detuned” static light scattering channel of a Wyatt DynaPro NanoStar light scattering instrument. A 10 μl volume of sample was loaded into a NanoStar disposable cuvette. The instrument was set to collect data with the laser power reduced to 1%. The static scattering detector voltage was recorded and calibrated against a set of polymer bead turbidity standards of 1, 5, 10, 20, 40, and 80 NTU.

[0377] Spray freeze drying

[0378] Spray freeze drying was performed using process parameters similar to those described by Clenet et al. (Clénet D, Hourquet V, Woinet B, Ponceblanc H, Vangelisti M. A spray freeze dried micropellet based formulation proof-of-concept for a yellow fever vaccine candidate. Eur J Pharm Biopharm. 2019 Sep; 142: 334-343). High level process parameters for spray freeze drying:

[0379]

[0380]

[0381] Spray drying

[0382] High level process parameters for spray drying:

[0383] Temperature(℃) 90 Temperature Outdoor(℃) Approx. 54 Temperature before cycle (℃) 40 <![CDATA[Air flow (m 3 / min)]]> 0.4 Cooling air (L / min) 300 Nozzle air (L / min) 8 <![CDATA[Absolute humidity (g / m 3 )]]> 10

[0384] Example 1: Feasibility of obtaining a high concentration formulation

[0385] Eight monoclonal antibodies (labeled as "mAb A", "mAb B", "mAb C", "mAb D", "mAb E", "mAb F", "mAb G" and "mAb H") were used for this feasibility assessment. Although mAb A, B and G are IgG4 subtypes, the others belong to the IgG1 subtype. All antibodies were formulated in a starting aqueous composition A consisting of 100 mg / mL antibody, 10 mM histidine, 2% trehalose, 0.05% polysorbate 80 (the ratio of the amount of antibody to the amount of trehalose was 10:2 w / w). The pH of each composition was selected based on isoelectric point and formulation screening studies. Each composition A was freeze-dried to produce an intermediate dried antibody powder (composition B). The intermediate dried antibody powder was reconstituted with an aqueous solution containing 1.75% or 100 mM arginine to obtain an aqueous antibody composition (composition I). The reconstitution volumes were selected to obtain aqueous antibody compositions I at 100, 150, 200, 250, 300, 350 and 400 mg / mL antibody concentrations. All eight antibodies achieved target protein concentrations as measured by SoloVPE. Clear and transparent solutions were generally obtained for all concentrations and antibodies tested ( Figure 1 ). Clear and transparent solutions up to 300 mg / mL were obtained for all eight antibodies. Clear and transparent solutions up to 350 mg / mL were obtained for seven antibodies, except mAb C. Clear and transparent solutions up to 400 mg / mL were obtained for mAb D, mAb G, and mAb H. Increases in antibody concentration are typically associated with increases in HMWS due to molecular crowding phenomena. However, in this example, increases in antibody concentration from 100 to 350 mg / mL were generally associated with marginal increases in HMWS (less than 1%) and moderate decreases in solution opalescence ( Figure 2 ). HMWS levels below 5% and HMWS changes of less than 1% with increasing concentrations are both robust indicators of physical stability of antibody composition I at an antibody concentration of at least 200 mg / mL. The opalescence of antibody composition I at all antibody concentrations of at least 200 mg / mL was less than 20 NTU, which is another robust indicator of optimal solution properties. Antibody composition I between 200 (inclusive) and 350 mg / mL (inclusive) provides optimal properties, such as clear and transparent solutions, low HMWS and opalescence.

[0386] Example 2: Optimal composition of aqueous composition A

[0387] Four monoclonal antibodies (mAb A, B, D and E) and eleven compositions were evaluated in this example. Table 1 provides an overview.

[0388] Table 1. Description of the 11 compositions of Example 2 evaluated using mAbs A, B, D, and E.

[0389]

[0390]

[0391] All antibodies are formulated in a starting aqueous composition A consisting of 100 mg / mL antibody, 10 mM histidine, 0.05% polysorbate 80, 0.5% to 4% trehalose (molecular weight 343), 0.45% to 3.5% arginine (molecular weight 174.2 g / mol). Trehalose is used as a cryoprotectant. This means that the ratio of the sum of the amount of antibody to the amount of cryoprotectant trehalose and arginine free alkali is 11:1 to 11:8%, and covers different combinations of trehalose and arginine between 0.5% to 4% and 0.45% to 3.5%, respectively. The pH of these formulations is antibody-specific and is selected during formulation screening to provide optimal stability. The antibodies in the starting aqueous composition A are freeze-dried to produce an intermediate dried antibody powder (composition B). The results are shown in Table 2.

[0392] Table 2. Evaluation of the physical stability of Composition A during freeze drying

[0393]

[0394] As shown in Table 2, for all antibodies and compositions, there was only a marginal increase in HMWS during freeze drying (<0.7% in all cases), which generally indicates that all antibody / trehalose + arginine compositions between 11:1 and 11:8 w / w protected the antibodies from degradation during freeze drying. An increase of 0.7% in HMWS is generally a robust indicator that composition A has stability during freeze drying. The data also indicate that higher levels of trehalose and arginine (e.g., 11:2 to 11:8) compositions are generally beneficial for antibody stability. We also observed that relative to trehalose alone ( Figure 3 The composition contains at least 0.45% arginine (MW 174.2 g / mol) ( Figure 3 The starting aqueous composition A (black bars in the middle) generally had a lower increase in HMWS during freeze drying. Evaluation of the short-term storage stability of the intermediate freeze-dried antibodies (composition B) at 25°C showed a modest increase in HMWS (typically <1% for mAb A, D, E, and typically <2% for mAb B) when stored at 25°C for 1 month relative to the initial time point, generally indicating that all compositions provided protection during storage of the intermediate freeze-dried antibodies (Table 3).

[0395] Table 3. Evaluation of the physical stability of composition B after storage at 25°C for 1 month

[0396]

[0397] The data also indicate that higher levels of trehalose and arginine (e.g., 11:2 to 11:8) compositions generally favor antibody stability during storage at 25°C. Evaluation of the accelerated stability of the intermediate freeze-dried antibody (Composition B) at 40°C indicated that relative to trehalose alone ( Figure 4 The composition of the present invention comprises a gray bar in the middle of the composition, a composition comprising at least 0.45% arginine (MW 174.2 g / mol) ( Figure 4 The black bars in the middle (composition B) generally have a lower HMWS increase. Subsequently, the intermediate dried antibody powder (composition B) is reconstituted with water for injection (WFI). The reconstitution volume is selected to obtain an aqueous antibody composition I (composition I) at a concentration of 300 mg / mL antibody. Figure 5 All antibodies and compositions shown in the obtained target protein concentration of 300mg / mL of clear and transparent aqueous solution. 300mg / mL antibody composition I in comparison with the HMWS in the 100mg / mL starting aqueous antibody composition A (both have the same antibody to trehalose, arginine weight ratio) generally indicates a marginal increase in HMWS due to a three-fold increase in antibody concentration. For mAb A, D and E, the HMWS change between antibody composition I and antibody composition A was less than 0.5%. mAb B showed a substantial increase in HMWS (less than 4%), but this increase was generally slowed down at 11: 2 to 11: 8w / w ( Figure 6 ), where the change in HMWS was generally less than 2%. Evaluation of the accelerated stability of 300 mg / mL antibody composition I showed a modest increase in HMWS (<2%) at 40°C for 2 weeks relative to the initial time point, which generally indicates that all compositions provide protection for the antibodies in composition I ( Fig.10 ). 40°C represents standard conditions for accelerated testing of biologics and is not the expected storage temperature for Composition I. An increase in HMWS of <2% at 40°C for 2 weeks is considered a small change and generally indicates high physical stability.

[0398] Example 3: Optimal reconstitution solution for intermediate dried antibody powder

[0399] Six monoclonal antibodies (labeled as mAb A, B, D, E, G and H) were used for this evaluation. All antibodies were formulated in a starting aqueous composition A consisting of 100 mg / mL antibody, 10 mM histidine, 2% trehalose, 0.05% polysorbate 80 (representing approximately 11:2 w / w) (Formulations F12 to F15) or 100 mg / mL antibody, 10 mM histidine, 3% trehalose, 0.05% polysorbate 80 (representing approximately 11:3 w / w) (Formulations 16 to 18). The antibodies in the starting aqueous composition A were freeze-dried to produce an intermediate dried antibody powder (Composition B). Subsequently, the dried antibody powder was reconstituted with 1.5% or 200mM glycine (F12), 3% or 150mM arginine-Cl (F13), 6.4% or 200mM arginine-glutamate (F14), 5.8% or 150mM magnesium-glutamate (F15), 3% or 150mM arginine-Cl (F16), 3% or 150mM arginine-Cl and 1.5% or 200mM glycine (F17), or 3% or 150mM arginine-Cl and 5.8% or 150mM magnesium-glutamate (F17). The reconstitution volume was selected to obtain an aqueous antibody composition I at a concentration of 300mg / mL of antibody. All antibodies and compositions obtained a clear and transparent aqueous solution ( Figure 8 ). Evaluation of accelerated stability of 300 mg / mL antibody composition I showed a modest increase in HMWS (<2%) at 40°C at 2 weeks relative to the initial time point, generally indicating that all compositions provided protection for the antibodies in composition I ( Fig. 9 ). 40°C represents standard conditions for accelerated testing of biologics and is not the expected storage temperature for Composition I. An increase in HMWS of <2% at 40°C for 2 weeks is considered a small change and generally indicates high physical stability. These changes in HMWS are consistent with Figure 4 The observations in (which involve trehalose and arginine-Cl) are similar. This generally indicates that the reconstitution solution containing glycine, arginine-Cl, guanidine-Cl, arginine-glutamate and magnesium-glutamate effectively provides protection for the antibodies in composition I.

[0400] Example 4: Evaluation of spray drying and spray freeze drying to produce intermediate dried antibody powders

[0401] Two monoclonal antibodies (labeled mAb B and I) and two compositions of each antibody (F20 and F21 for mAb I, F22 and F23 for mAb B) were used for this evaluation. The starting aqueous composition A for mAb B consisted of (F22) 100 mg / mL antibody, 0.06% polysorbate 80, 1.75% arginine (representing approximately 11:2 w / w antibody:trehalose+arginine) and (F23) 100 mg / mL antibody, 0.06% polysorbate 80, 2% trehalose, 0.9% arginine (representing approximately 11:3 w / w antibody:trehalose+arginine). The starting aqueous composition A of mAb I consisted of (F20) 100 mg / mL antibody, 10 mM histidine, 0.06% polysorbate 80, 3% trehalose, 0.9% arginine (representing approximately 11:4 w / w) and (F21) 100 mg / mL antibody, 10 mM histidine, 0.06% polysorbate 80, 2% trehalose, 0.45% arginine (representing approximately 11:2.5 w / w). The two antibodies in their respective starting compositions A were spray dried and spray freeze dried to produce intermediate dried antibody powders (composition B). Fig.10 As shown, spray drying produces loose dry powders, and spray freeze drying produces micropills. The antibody powder (composition B) from the intermediate drying of the two drying techniques was reconstituted with water for injection. The reconstitution volume was selected to obtain an aqueous antibody composition I at a concentration of 300 mg / mL antibody. After the spray freeze-dried micropills were reconstituted, the antibodies (mAb B and I) and compositions (F22 and F23 for mAb B, F20 and F21 for mAb I) all obtained a clear and transparent aqueous solution ( Fig.11 ). For both compositions of mAb B, the opalescence of the resulting solutions was <5 NTU, generally indicating acceptable solution characteristics. The reconstitution time for reconstitution of the micropellets was typically 1 to 2 hours. Although antibody concentrations of 300 mg / mL were obtained after reconstitution of the spray-dried powder, the reconstitution time was typically greater than 4 hours, and the resulting solutions typically had high turbidity (>20 NTU) and generally lacked clarity and transparency. Evaluation of the short-term and long-term storage stability of the intermediate spray-dried powder and spray-freeze-dried micropellets (Composition B) generally showed no significant increase in HMWS after short-term storage at 25°C or long-term storage at 5°C ( Fig.12 Evaluation of the short-term storage and accelerated stability of 300 mg / mL Composition I produced by reconstitution of spray-dried powders and spray-freeze-dried micropellets generally showed no significant increase in HMWS after short-term storage at 5°C or 25°C for 3 months or at 40°C for 1 month ( Fig.13 ).

Claims

1. A method for producing an aqueous antibody composition I comprising at least 200 mg / ml antibody, wherein the method comprises the following steps: a) providing an aqueous composition A comprising a1) an antibody, the concentration of which is lower than the concentration of the antibody in the antibody composition I, a2) at least one surfactant, a3) at least one cryoprotectant, a4) at least 0.4% (w / v) arginine or a salt thereof, wherein the weight (w) is calculated as arginine, e.g. 0.4% to 3.5% (w / v) arginine, wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8, and b) drying the composition provided in step a) to produce antibody composition B, and c) reconstitution of the composition dried in step b), To give the aqueous antibody composition I.

2. The method of claim 1, wherein the salt of arginine is arginine-Cl, arginine-aspartate, arginine-glutamate, arginine-sulfate, arginine-acetate, arginine-succinate.

3. The method of claim 1 or 2, wherein the at least one cryoprotectant is selected from the group of cryoprotectants consisting of trehalose, sucrose, sorbitol, glycerol and mannitol.

4. The method of any one of claims 1 to 3, wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:4, such as 11:2 to 11:

4.

5. The method of any one of claims 1 to 4, wherein the aqueous composition A comprises less than 200 mg / ml antibody, such as 10 to 190 mg / ml antibody, such as 20 to 170 mg / ml antibody, such as 50 to 150 mg / ml antibody, such as 80 to 120 mg / ml antibody.

6. The method of any one of claims 1 to 5, wherein composition I comprises 230 mg / ml to 420 mg / ml antibody, such as 260 mg / ml to 400 mg / ml antibody, or Wherein Composition I comprises 270 to 350 mg / ml antibody, for example 280 to 320 mg / ml antibody, for example 300 mg / ml antibody.

7. The method according to any one of claims 1 to 6, wherein a) the antibody is a monoclonal antibody, b) the at least one surfactant is a polysorbate, such as polysorbate 20 or 80, or a poloxamer, such as poloxamer 188, c) the pH of the composition A is about 5.0 to 8.0, such as about 5.5 to 7, and / or d) Composition A further comprises a5) a buffer, such as a histidine, citrate, acetate, phosphate, Tris, succinate or glycine buffer.

8. The method according to any one of claims 1 to 7, wherein the antibody composition B is in the form of a powder or pellets.

9. The method of any one of claims 1 to 8, wherein step b) is performed by freeze drying or spray drying to give a freeze dried or spray dried powder, and optionally wherein the freeze dried powder is reconstituted in a reconstitution solution for a period of 1.5 to 4.0 hours, or wherein the spray dried powder is reconstituted in a reconstitution solution for a period of at least four hours.

10. The method according to any one of claims 1 to 8, wherein step b) is performed by spray freeze drying to give spray freeze dried micropellets.

11. The method of claim 10, wherein in step c), the spray freeze-dried micropellets are reconstituted in a reconstitution solution for a period of 1 to 2 hours.

12. An aqueous antibody composition I obtained or obtainable by the method according to any one of claims 1 to 11.

13. A container comprising the aqueous antibody composition I of claim 12, for example, wherein the container is a prefilled syringe, vial, cartridge, ampoule or autoinjector.

14. A method for producing a dried antibody composition B, a) providing an aqueous composition A comprising a1) less than 200 mg / ml antibody, such as 10 to 190 mg / ml antibody, such as 20 to 170 mg / ml antibody, such as 50 to 150 mg / ml antibody, such as 80 to 120 mg / ml antibody, a2) at least one surfactant, a3) at least one cryoprotectant, and a4) at least 0.4% (w / v) arginine or a salt thereof, wherein the weight (w) is calculated as arginine, e.g. 0.4% to 3.5% (w / v) arginine, wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:8, and b) spray freeze-drying the composition provided in step a), To give the dried antibody composition B.

15. The method of claim 14, wherein the dried antibody composition is in the form of micropellets.

16. The method of claim 14 or 15, wherein the salt of arginine is arginine-Cl, arginine-aspartate, arginine-glutamate, arginine-sulfate, arginine-acetate, arginine-succinate, for example wherein the salt of arginine is arginine-Cl.

17. The method of any one of claims 14 to 16, wherein the at least one cryoprotectant is selected from the group of cryoprotectants consisting of trehalose, sucrose, sorbitol, glycerol and mannitol, such as wherein the cryoprotectant is trehalose.

18. The method of any one of claims 14 to 17, wherein the ratio of the amount of a1) to the sum of the amounts of a3) and a4) is 11:1 to 11:4, such as 11:2 to 11:

4.

19. The method of any one of claims 14 to 18, wherein the aqueous composition A comprises 50 to 150 mg / ml antibody, such as 80 to 120 mg / ml antibody.

20. The method of any one of claims 14 to 19, wherein composition A further comprises a5) a buffer, such as a histidine, citrate, acetate, phosphate, Tris, succinate or glycine buffer.

21. The method of any one of claims 14 to 20, wherein the antibody is a monoclonal antibody.

22. A dried antibody composition B obtained or obtainable by the method according to any one of claims 14 to 21.

23. Use of a dried antibody composition B as claimed in any one of claims 14 to 22 or an aqueous antibody composition A as defined in any one of claims 1 to 21 for producing an aqueous antibody composition I comprising at least 200 mg / ml antibody.

24. An aqueous composition A as defined in any one of claims 1 to 21.

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