Method for preparing high concentration liquid formulations of antibodies

Abstract

The present invention relates to a method for preparing a high concentration liquid formulation of an antibody. The present invention provides a method for preparing a high concentration liquid formulation (HCLF) of an antibody or fragment thereof. The present invention also relates to a method for stabilizing an anti-CD20 antibody or fragment thereof in a liquid pharmaceutical formulation. Furthermore, the present invention relates to a liquid pharmaceutical formulation of a vistuzumab antibody or a fragment thereof, said liquid pharmaceutical formulation comprising at least 155 mg / mL of a vistuzumab antibody or a fragment thereof.

Description

[0001] The present application is a divisional application of the international application PCT / EP2013 / 072750 with an international application date of October 31, 2013, which entered into China with application number 201380056798.5 and is entitled “Method for Preparing High-Concentration Liquid Preparations of Antibodies”. Technical Field

[0002] The present invention provides a method for preparing a high concentration liquid formulation (HCLF) of an antibody. The present invention also relates to a method for stabilizing an anti-CD20 antibody or a fragment thereof in a liquid pharmaceutical formulation. In addition, the present invention relates to a liquid pharmaceutical formulation of a veltuzumab antibody or a fragment thereof and the use of the pharmaceutical formulation as a medicament. Background Art

[0003] introduction Therapeutic proteins must meet many different criteria, e.g. regarding stability, dosing and concentration, to meet regulatory approval requirements as drugs. During the manufacturing process, chemical and / or physical degradation of therapeutic proteins such as antibodies, storage and transport may occur, which can lead to a loss of their pharmaceutical efficacy and an increased risk of side effects (e.g., unwanted immune responses).

[0004] Many therapeutic proteins need to be administered in high doses to achieve their desired therapeutic effect. In addition, high concentration formulations of therapeutic proteins are advantageous because they can allow for a more convenient mode of administering the therapeutic protein to a patient.

[0005] High concentrations (e.g., at least 100 mg / mL) of therapeutic proteins are desirable because as concentration increases, the volume required to administer a therapeutic dose decreases. Smaller volumes provide the advantage that they can be injected via less invasive routes, such as subcutaneous injection instead of intravenous infusion, which is more convenient for patients and potentially associated with less risk of side effects (e.g., infusion reactions). Another advantage provided by high concentration formulations is that they can allow for a reduction in the frequency of administering therapeutic proteins to patients.

[0006] Then, it is challenging to provide highly concentrated liquid preparations of therapeutic proteins (such as monoclonal antibodies) because the viscosity of the liquid preparations and the tendency of the protein to form aggregates can be significantly increased at higher concentrations. Aggregates may contain degradation products of proteins and may cause unwanted side effects, such as triggering unwanted immune responses. In order to avoid stability problems such as forming aggregates, freeze drying can be used. Therefore, many approved products with a concentration higher than 100 mg / mL are lyophilized products (lyophilisate), which must be reconstructed before use. However, freeze drying is a time-consuming and expensive process. In addition, the reconstruction of lyophilized products is inconvenient and error-prone for patients and medicinal individuals.

[0007] A number of documents discuss highly concentrated antibody preparations and / or methods for providing the same: WO03 / 039485 describes stable liquid pharmaceutical formulations of antibodies having an antibody concentration of 50 mg / mL or greater, in particular Daclizumab (anti-IL2 receptor antibody), HAIL-12 (humanized anti-IL12 antibody), HuEP5C7 (humanized anti-selectin monoclonal antibody) and Flintozumab (humanized anti-γ interferon monoclonal antibody). However, neither the high-concentration liquid formulation of the Veltuzumab antibody according to the present invention nor the method for providing the high-concentration liquid formulation of the antibody is disclosed therein.

[0008] US2012 / 0064086 also describes highly concentrated antibody preparations, in particular HCLF of IgE antibodies, which HCLF has a reduced viscosity. However, said document does not disclose an antibody preparation comprising veltuzumab antibody as described herein or a method for preparing a highly concentrated liquid preparation of an antibody.

[0009] WO 2011 / 029892 relates to highly concentrated, stable anti-CD20 antibody formulations. However, it does not describe highly concentrated formulations of the veltuzumab antibody as disclosed herein or methods for preparing HCLF.

[0010] WO 2004 / 001007 describes a method for providing a composition of an antibody consisting essentially of an antibody and an aqueous solution of histidine or acetate buffer, in the range of 2 mM to 48 mM. However, the method disclosed therein does not include a step of ultrafiltration up to about 280 mg / mL.

[0011] US2012 / 0064086 also discloses a method for producing a highly concentrated antibody formulation with reduced viscosity, the method comprising three filtration steps (ie, a first ultrafiltration step, a diafiltration step, and a second ultrafiltration step) performed at high temperature.

[0012] More general considerations for developing protein pharmaceutical formulations are described in the mini-review “Challenges in the development of high protein concentration formulations” (Steven J. Shire, Zahra Shahrokh, Jun Liu Journal of Pharmaceutical Sciences, Vol. 93, No. 6, June 2004, 1390-1402).

[0013] Therefore, there exists a need for high concentration liquid formulations of antibodies and methods of providing such high concentration liquid formulations of antibodies. Summary of the invention

[0014] One aspect of the present invention relates to a method for preparing an antibody having a concentration C H A method for preparing a high concentration liquid formulation of an antibody, comprising the steps of: a) provide a starting concentration of C S A solution of an antibody; b) ultrafiltration of the solution of step (a) to obtain a solution having an intermediate antibody concentration C I A solution of which C I is at least about 260 mg / mL; and c) diluting the solution of step (b) to an antibody concentration of C H To obtain a high concentration liquid preparation.

[0015] One embodiment of the present invention relates to a process for preparing HCLF according to the present invention, wherein the solution of step (a) further contains a buffer, which is optionally an amino acid, such as histidine, in particular L-histidine.

[0016] Another embodiment of the present invention relates to a process for preparing HCLF according to the present invention, said process further comprising a step of diafiltration of the solution of step (a) with a buffer solution between step (a) and step (b), wherein the buffer is optionally an amino acid, such as histidine, in particular L-histidine.

[0017] Another embodiment of the present invention relates to a process for preparing HCLF according to the present invention, wherein the solution subjected to ultrafiltration in step (b) contains 40 mM histidine and has a pH value of 5.45.

[0018] One embodiment of the present invention relates to a process for the preparation of HCLF according to the present invention, wherein the solution subjected to ultrafiltration in step (b) contains substantially no or no tonicity modifier, such as sucrose.

[0019] Another embodiment of the present invention relates to a process for the preparation of HCLF according to the present invention, wherein the solution subjected to ultrafiltration in step (b) contains substantially no or no surfactant, such as polysorbate.

[0020] Another embodiment of the present invention relates to a method for preparing HCLF according to the present invention, wherein the antibody is an anti-CD20 antibody and / or an IgG antibody, optionally the antibody is a veltuzumab antibody.

[0021] One embodiment of the present invention relates to a method for preparing HCLF according to the present invention, wherein the high concentration liquid formulation has an antibody concentration C of at least 155 mg / mL. H .

[0022] One embodiment of the present invention relates to a process for preparing HCLF according to the present invention, wherein step (b) is carried out in an ultrafiltration device and further comprises a step of flushing the ultrafiltration device with a buffer solution between step (b) and step (c), wherein the buffer is optionally an amino acid, such as histidine, in particular L-histidine.

[0023] Another aspect of the invention relates to a method for stabilizing an anti-CD20 antibody or fragment thereof at a concentration of at least 155 mg / mL in a liquid pharmaceutical formulation by mixing the non-lyophilized antibody or fragment thereof with an aqueous solution comprising an amino acid, optionally histidine, particularly L-histidine.

[0024] One embodiment of the present invention relates to a method for stabilizing an anti-CD 20 antibody or a fragment thereof, wherein the anti-CD20 antibody is a veltuzumab antibody.

[0025] Another aspect of the present invention relates to a liquid pharmaceutical formulation of a veltuzumab antibody or a fragment thereof, comprising at least 155 mg / mL of a veltuzumab antibody or a fragment thereof and an amino acid, wherein the amino acid is optionally histidine, in particular L-histidine.

[0026] One embodiment of the present invention relates to a liquid pharmaceutical formulation, wherein the concentration of the veltuzumab antibody or fragment thereof is at least 175 mg / mL, preferably at least 190 mg / mL.

[0027] Another embodiment of the present invention relates to a liquid pharmaceutical formulation, wherein the concentration of the veltuzumab antibody or fragment thereof is at least 200 mg / mL, preferably at least 220 mg / mL.

[0028] Another embodiment of the present invention relates to a liquid pharmaceutical formulation, wherein the concentration of the amino acid, optionally histidine, in particular L-histidine, is in the range of 1 mM to 100 mM.

[0029] Another embodiment of the present invention relates to a liquid pharmaceutical formulation, wherein the concentration of the amino acid, optionally histidine, in particular L-histidine, is in the range of 10 mM to 60 mM, optionally in the range of 20 mM to 50 mM.

[0030] One embodiment of the present invention relates to a liquid pharmaceutical formulation further comprising a surfactant, optionally a non-ionic surfactant.

[0031] One embodiment of the present invention relates to a liquid pharmaceutical formulation, wherein the surfactant (optionally a non-ionic surfactant) is a polysorbate, optionally polysorbate 20 or polysorbate 80.

[0032] Another embodiment of the present invention is directed to a liquid pharmaceutical formulation, wherein the surfactant, optionally a nonionic surfactant, is present in a concentration of at least 0.01 mg / mL, optionally in the range of 0.1 mg / mL to 1 mg / mL.

[0033] One embodiment of the present invention relates to a liquid pharmaceutical formulation further comprising a tonicity modifier.

[0034] Another embodiment of the present invention relates to a liquid pharmaceutical formulation, wherein the tonicity modifier is sorbitol and / or mannitol, optionally the concentration of the tonicity modifier is in the range of 5.5 mM to 550 mM.

[0035] One embodiment of the present invention relates to a liquid pharmaceutical formulation having a pH value in the range of 4.8 to 7.0, optionally a pH value of 5.5±0.3.

[0036] One embodiment of the present invention relates to a liquid pharmaceutical preparation comprising a) at least 160 mg / mL of veltuzumab antibody or a fragment thereof; b) 220 mM sorbitol; c) 30 mM histidine; d) 0.2 mg / mL polysorbate 20; and Having a pH value in the range of 5.0 to 6.0, optionally 5.5±0.3.

[0037] Another embodiment of the present invention relates to a liquid pharmaceutical formulation for use as a medicament.

[0038] Another embodiment of the present invention is directed to a liquid pharmaceutical formulation, wherein the medicament is for subcutaneous administration.

[0039] One embodiment of the present invention relates to a liquid pharmaceutical formulation for use in treating cancer or a non-malignant disease, optionally an inflammatory or autoimmune disease, including a Class III autoimmune disease.

[0040] Another embodiment of the present invention relates to a liquid pharmaceutical formulation for use in treating a disease selected from the group consisting of Burkitt's lymphoma, Epstein-Barr virus infection, B-cell leukemia, chronic lymphocytic B-cell leukemia, acute lymphocytic leukemia, lymphatic leukemia, prolymphocytic leukemia, hairy cell leukemia, multiple myeloma, B-cell lymphoma, marginal zone B-cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, immunoblastic large cell lymphoma, mantle cell lymphoma, non-Hodgkin's lymphoma, lymphomatoid granuloma, plasmacytoma, precursor cell lymphocytic leukemia-lymphoma, tumor virus infection, Waldenstrom's macroglobulinemia, immunoproliferative disorders, prolymphocytic lymphoma, diffuse large B-cell lymphoma, immunoblastic large cell lymphoma, mantle cell lymphoma, lymph leukemia, leukemia-derived lymphoma, thrombocytopenic purpura, idiopathic thrombocytopenic purpura, blood coagulation disorders, platelet diseases, hemoproteinopathies, blood diseases, bleeding disorders, coagulation disorders, lymphatic diseases, purpura, thrombocytopenia, thrombotic microangiopathy, coagulation disorders, vascular diseases, systemic lupus erythematosus (SLE), lupus (e.g., lupus nephritis, non-renal lupus, discoid lupus, lupus alopecia) (Lupus (egnephritis, non-renal, discoid, alopecia)), juvenile onset diabetes, multiple sclerosis, rheumatoid arthritis, rheumatic diseases, connective tissue diseases, herpes virus infections and / or DNA virus infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1An exemplary method for providing HCLF without an upstream diafiltration (DF) step according to the present invention is shown. The final product in this example has a pH of 5.5 and contains 30 mM histidine (His). To counteract the Donnan effect during ultrafiltration (UF), a higher histidine concentration and a lower pH value can be adjusted in the starting material. Due to the so-called Donnan effect, charged molecules that can pass through the UF membrane are depleted (e.g., in this example) or concentrated in the solution, depending on the charge sign related to the charge of the protein that cannot pass through the membrane. The depletion of positively charged histidine is accompanied by a change in solution pH. A bulk drug substance containing 5 g / L veltuzumab and 40 mM histidine and having a pH of 5.45 can be used as a starting material. The starting material is subjected to an ultrafiltration step. In the ultrafiltration step, veltuzumab is concentrated to a concentration of up to about 285 g / L. After reaching this concentration, the ultrafiltration device can be rinsed with a diafiltration buffer containing 40 mM histidine and having a pH of 5.45. This can maximize the yield of the antibody obtained by the method described herein. The drug product pool thus obtained in this example has a concentration of 195g / L veltuzumab, a pH value of 5.5 and comprises approximately 30mM histidine. Subsequently, a further dilution step can be carried out using a so-called 10-fold polysorbate-sorbitol spike buffer solution comprising 30mM histidine, 2.2M sorbitol, 2g / L polysorbate and having a pH value of 5.5. The polysorbate-sorbitol spike buffer solution comprises 10 times the concentration of the desired concentration of polysorbate and sorbitol in the final product. The resulting drug product mixture in this example comprises 175g / L veltuzumab, 30mM histidine, 220mM sorbitol, 0.2g / L polysorbate 20 and has a pH value of 5.5. Then, the drug product can be finally adjusted to the desired concentration by diluting with a so-called 1-fold formulation buffer solution containing 30 mM histidine, 220 mM sorbitol and 0.2 g / L polysorbate 20 and having a pH value of 5.5. The formulation buffer solution contains polysorbates and sorbitol concentrations similar to the final drug product mixture (i.e., 1-fold concentration). The final drug product mixture in this example contains 160 g / L veltuzumab, 30 mM histidine, 220 mM sorbitol and 0.2 g / L polysorbate 20 and has a pH value of 5.5.

[0042] Figure 2 Another exemplary method of the present invention disclosed in Figure 1The method disclosed in is different in that the starting material contains 5 g / L veltuzumab and 10 mM histidine and has a pH of 5.5. The ultrafiltration is preceded by a diafiltration step in which the final solution is balanced by removing the pre-buffer composition by exchanging 8 volumes of the buffer with a diafiltration buffer solution containing 40 mM histidine and having a pH of 5.45. The subsequent steps are the same as those Figure 1 The steps performed in the exemplary methods shown are consistent.

[0043] Figure 3 The exemplary methods of the present invention disclosed in Figure 1 The method disclosed in is different in that the starting material comprises 60 g / L veltuzumab, 120 mM sucrose and 10 mM histidine and has a pH of 5.5. A diafiltration step is performed before ultrafiltration. Sucrose is thereby removed. The buffer is then exchanged 8 times by using a diafiltration buffer solution comprising 40 mM histidine and having a pH of 5.45. The steps subsequently performed are the same as Figure 1 The steps performed in the method shown are identical.

[0044] Figure 4 Response Contour Plot showing formulation optimization; aggregation (expressed as % of absolute antibody amount) after six months at 2-8°C. DETAILED DESCRIPTION

[0045] definition If the term "comprise" or "comprising" is used in the present description and claims, it does not exclude other elements or steps. For the purpose of the present invention, the term "consisting of" is considered as an optional embodiment of the term "comprising". If hereinafter a group is defined as comprising at least a certain number of embodiments, this is also understood to disclose a group optionally consisting of only these embodiments.

[0046] Where an indefinite or definite article is used when referring to a singular noun eg "a" or "an", "the", this includes a plural of that noun unless something else is said.

[0047] Vice versa, when a noun is used in the plural, it also refers to the singular. For example, when referring to anti-CD20 antibodies, this is also understood to refer to a single anti-CD20 antibody.

[0048] In addition, the terms first, second, third or (a), (b), (c), etc. in the specification and claims are used to distinguish similar elements and are not necessarily used to describe sequential or chronological order. It should be understood that the terms used in this way are interchangeable where appropriate and that the embodiments of the present invention described herein can be operated in other sequences different from those described or illustrated herein. However, in a specific embodiment of the present invention, method steps (a), (b) and (c) (optionally including any intermediate steps defined herein) are performed in chronological order.

[0049] In the context of the present invention, any numerical value indicated is generally associated with an interval of accuracy, wherein a person skilled in the art will understand that it still ensures the technical effect of the feature in question. As used herein, the deviations of the numerical values ​​indicated are within the range of ±10%, and preferably within the range of ±5%. The aforementioned deviations of ±10% and preferably ±5% of the numerical interval indicated are also represented by the terms "about" and "approximately" used herein with respect to numerical values.

[0050] As used herein, "substantially free" means that the identified component is not present in an amount that can be detected under typical conditions for its detection. In addition, "substantially free" also includes that the component is not present in an amount that adversely affects the desired properties of the composition or formulation, that is, the identified compound is present in a negligible amount.

[0051] As used herein, the term "liquid preparation" refers to a liquid preparation and includes liquid preparations that were originally liquid and resuspended lyophilized / freeze-dried preparations, i.e., so-called reconstituted solutions. However, it is not necessary for stability reasons that the liquid preparations disclosed herein are lyophilized / freeze-dried or have a state that is changed by other methods (e.g., by spray drying). In one embodiment of the invention, the term "liquid preparation" does not include resuspended or reconstructed lyophilized / freeze-dried preparations.

[0052] In the context of the present invention, the term "antibody" refers to full-length antibodies, human antibodies, humanized antibodies, fully human antibodies, genetically engineered antibodies (e.g., monoclonal antibodies, polyclonal antibodies, chimeric antibodies, recombinant antibodies) and multispecific antibodies, as well as fragments of such antibodies that retain the characteristic properties of full-length antibodies. In one embodiment, the antibody is a humanized antibody. A "humanized antibody" is an antibody that has been modified to provide an increased similarity to an antibody produced by humans, for example by grafting mouse CDRs into the framework region of a human antibody.

[0053] The term "antibody fragment" refers to a portion of a full-length antibody that binds to the same antigen as the full-length antibody. In particular, it refers to a pharmaceutically active fragment of an antibody. This portion of a full-length antibody may be at least the antigen binding portion or at least its variable region. Genetically engineered proteins that function like antibodies are also included within the meaning of antibody fragments as used herein. Such genetically engineered antibodies may be scFv, i.e., a fusion protein in which the heavy and light chain variable regions are connected by a peptide linker. Other exemplary antibody fragments are Fab, Fab', F(ab')2, and Fv.

[0054] As defined herein, an anti-CD 20 antibody refers to any antibody or fragment thereof that specifically binds to the CD 20 antigen (also known as CD 20 (cluster of differentiation antigen 20), B-lymphocyte antigen CD 20, B-lymphocyte surface antigen B1, Leu-16 or Bp35). This includes anti-CD 20 antibodies with marketing approval, anti-CD 20 antibodies currently being investigated in clinical trials and / or any other compound that specifically binds to the CD 20 antigen. As used herein, the CD 20 antigen relates to any variant, subtype and species homolog of human CD 20. As used herein, the anti-CD 20 antibody refers to type I anti-CD 20 antibodies as well as type II anti-CD 20 antibodies, which differ in the mode of CD 20 binding and biological activity. Examples of anti-CD 20 antibodies are veltuzumab, rituximab, ocrelizumab, ofatumumab, Y 90 Ibritumomab tiuxetan 131 Tositumomab, TRU-015, AME-133v, humanized PRO131921, GA101, 1F5 IgG2a, HI47 IgG3, 2C6IgG1, 2H7 IgG1, AT80IgG1, 11B8 IgG1, humanized B-Ly1 antibody IgG1, and Aftuzumab (HuMab <cd20>). In particular, the anti-CD20 antibody is veltuzumab or a fragment thereof.

[0055] Veltuzumab is a monoclonal humanized anti-CD 20 antibody of the IgG1 / κ class consisting of mature heavy and light chains of 451 and 213 amino acid residues, respectively. Veltuzumab has the amino acid sequence shown below:

[0056] The anti-CD20 antibodies according to the present invention can be monoclonal or polyclonal antibodies. Monoclonal antibodies are monospecific antibodies (i.e., bound to the same epitope) derived from a single cell line. Therefore, monoclonal antibodies are substantially identical antibodies except for variants that occur during their production. In contrast, polyclonal antibodies relate to a variety of antibodies directed against different antigenic epitopes. Methods for producing monoclonal and polyclonal antibodies are known in the art and include, for example, hybridoma technology and recombinant DNA methods. In one embodiment of the invention, the anti-CD20 antibody is a monoclonal antibody.

[0057] According to the present invention, "IgG antibody" refers to a therapeutically useful antibody or fragment thereof that falls within the scope of IgG class (isotype) antibodies and has a gamma-type (γ) heavy chain. This includes antibodies of any subtype of the IgG class known in the art, i.e., IgG1, IgG2, IgG3 or IgG4. In one embodiment, the antibody is an IgG1 antibody. It should be understood herein that IgG antibodies also include antibodies that specifically bind to the CD 20 antigen, and vice versa. An exemplary IgG antibody of the present invention is veltuzumab.

[0058] It is understood that "binds specifically or specifically binds" refers to a binding affinity of ≤10 -9 mol / l, especially ≤10 -10 mol / l of an antibody having a binding affinity for the CD 20 antigen as defined herein. Methods for determining the binding affinity of an antibody to an antigen are known in the art and include, for example, the use of surface plasmon resonance.

[0059] As used herein, "surfactant" refers to a pharmaceutically acceptable surface active agent. Surfactants can protect therapeutic proteins (e.g., antibodies as defined herein) from such interfacial tensions between two liquids or between a liquid and a solid and / or can reduce the tendency to aggregate or form particles. Pharmaceutically acceptable surfactants include nonionic surfactants, such as polysorbates and poloxamers, but are not limited thereto. Exemplary surfactants useful in the present invention are polyoxyethylene-polyoxypropylene copolymers (e.g., poloxamer 188), polyoxyethylene alkyl ethers, and polysorbates (e.g., polysorbate 20, polysorbate 80). It should be understood that combinations of surfactants, such as combinations of the aforementioned surfactants, can also be used.

[0060] In the context of the present invention, "pharmaceutically acceptable" relates to any compound that can be used in a pharmaceutical composition without causing any undesirable effects (such as adverse side effects) in the patient to whom the composition is administered.

[0061] As used herein, "tonicity modifier" refers to any pharmaceutically acceptable agent suitable for providing an isotonic formulation. An isotonic formulation is a formulation having the same tonicity (i.e., solute concentration) as a compared formulation (e.g., whole blood, serum, or physiological saline solution). Suitable tonicity modifiers within the meaning of the present invention include NaCl, potassium chloride, glycine, glycerol, salts, amino acids, sugar alcohols (e.g., sorbitol and mannitol) and sugars (e.g., glucose, sucrose, trehalose, and glucose). Optionally, the tonicity modifier is a sugar or a sugar alcohol, in particular sorbitol, sucrose, and / or mannitol. In a specific embodiment, the tonicity modifier is a sugar, in particular sorbitol. It should be understood that a combination of tonicity modifiers, in particular a combination of the above-mentioned tonicity modifiers, may also be used. In particular, it should be understood that a tonicity modifier may be used to provide physiological tonicity in a formulation (i.e., a formulation having substantially the same tonicity as human blood). Such formulations will typically have an osmotic pressure concentration (osmolarity) of about 300mOsm / kg, in particular 310mOsm / kg.

[0062] The term "histidine" (His) as used herein specifically includes L-histidine, unless otherwise specified.

[0063] "Patient" as used herein relates to any mammal, especially a human, suffering from, diagnosed with, susceptible to or susceptible to any disease or condition mentioned herein.

[0064] According to the present invention, "treatment" or "therapy" relates to the therapeutic and / or prophylactic treatment of a patient as defined herein.

[0065] As used herein, "room temperature" means a temperature in the range of 18° C. to 25° C., especially in the range of about 19° C. to about 22° C. In particular, room temperature means 20° C. or 25° C.

[0066] Further definitions of the terms are given below in the context in which the terms are used. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for preparing a high concentration liquid formulation (HCLF) of an antibody or a fragment thereof. The method according to the present invention allows providing a HCLF and obtaining a relatively high yield of the antibody.

[0068] In one embodiment thereof, the HCLF has an antibody or fragment thereof concentration of at least 150 mg / mL, at least 155 mg / mL, at least 160 mg / mL, at least 175 mg / mL, at least 180 mg / mL, at least 185 mg / mL, at least 190 mg / mL, at least 195 mg / mL, at least 200 mg / mL, at least 220 mg / mL, at least 240 mg / mL, at least 260 mg / mL, at least 280 mg / mL, at least 285 mg / mL, or at least 290 mg / mL. H In a specific embodiment, the HCLF has an antibody concentration of at least 155 mg / mL. H In another specific embodiment, the HCLF has an antibody or fragment thereof concentration of at least 160 mg / mL. H In another specific embodiment, the HCLF has an antibody or fragment thereof concentration of at least 175 mg / mL. H In another specific embodiment, the HCLF has an antibody or fragment thereof concentration of at least 200 mg / mL. H In another embodiment of the present invention, the HCLF of the present invention has an antibody or fragment concentration C of 160-290 mg / mL, 160-250 mg / mL, 160-220 mg / mL, 160-200 mg / mL or 160-175 mg / mL. H .

[0069] The present invention provides a method for preparing a method having an antibody concentration C H A method for preparing a high concentration liquid formulation of an antibody or a fragment thereof, comprising the steps of: a) provide a starting concentration of C S A solution of an antibody; b) ultrafiltration of the solution of step (a) to obtain a solution having an intermediate antibody concentration C I A solution of which C I is at least about 260 mg / mL; and c) diluting the solution of step (b) to an antibody concentration of C H To obtain a high concentration liquid preparation.

[0070] In a specific embodiment, the method is used to prepare the HCLF of anti-CD 20 antibody and / or IgG antibody. In another specific embodiment, the method is used to prepare the HCLF of the combination of anti-CD 20 antibody and / or anti-CD 20 antibody and other active ingredients. In another specific embodiment, the method is used to prepare the HCLF of the combination of humanized monoclonal anti-CD 20 antibody and / or humanized monoclonal anti-CD 20 antibody and other active ingredients. In a specific embodiment, the method is used to prepare the HCLF of the combination of IgG antibody and / or IgG antibody and other active ingredients. In another embodiment of the present invention, the method is used to prepare the HCLF of the combination of veltuzumab antibody and / or veltuzumab antibody and other active ingredients.

[0071] In a specific embodiment, the method is used to prepare the HCLF of anti-CD 20 antibody fragments and / or IgG antibody fragments. In another specific embodiment, the method is used to prepare the HCLF of the combination of anti-CD 20 antibody fragments and / or anti-CD 20 antibody fragments and other active ingredients. In another specific embodiment, the method is used to prepare the HCLF of the combination of humanized monoclonal anti-CD 20 antibody fragments and / or humanized monoclonal anti-CD 20 antibody fragments and other active ingredients. In a specific embodiment, the method is used to prepare the HCLF of the combination of IgG antibody fragments and / or IgG antibody fragments and other therapeutic agents. In another embodiment of the present invention, the method is used to prepare the HCLF of the combination of veltuzumab antibody fragments and / or veltuzumab antibody fragments and other therapeutic agents.

[0072] Other therapeutic agents may be administered separately, simultaneously or sequentially with the formulation according to the invention. The other therapeutic agent may be any therapeutic agent suitable for treating any disease mentioned herein. Examples of such other therapeutic agents are, for example, cytotoxic agents, anti-angiogenic agents, corticosteroids, antibodies, anti-inflammatory agents, chemotherapy, hormones and immunomodulators.

[0073] Examples of such therapeutic agents may be selected from antimitotic agents, antikinase agents, alkylating agents, antimetabolites, antibiotics, alkaloids, antiangiogenic agents, apoptotic agents, and combinations thereof. Chemotherapeutic agents may be selected, for example, from drugs such as vinca alkaloids, anthracyclines, epipodophyllotoxins, taxanes, antimetabolites, alkylating agents, antikinase agents, antibiotics, Cox-2 inhibitors, antimitotic agents, antiangiogenic agents, and apoptotic agents, particularly doxorubicin, methotrexate, paclitaxel, CPT-II, camptothecin, and other drugs from these and other classes of anticancer agents.

[0074] Further examples of such therapeutic agents are described, for example, in International Patent Application WO 2003 / 068821, the contents of which are incorporated herein and expressly referenced.

[0075] In one embodiment of the invention, the starting concentration C of the antibody or fragment thereof as defined herein is S represents an antibody concentration of at least 2 g / L, at least 5 g / L, at least 10 g / L, at least 20 g / L, at least 30 g / L, at least 40 g / L, at least 50 g / L, at least 60 g / L, at least 70 g / L, at least 80 g / L, at least 90 g / L or at least 100 g / L. In one embodiment, the C of the antibody or fragment thereof in the solution provided in step a) is S In another embodiment, the C of the antibody or fragment thereof in the solution provided in step a) is at least 5 g / L. S At least 60 g / L.

[0076] In one embodiment of the invention, the solution provided in step a) of the method according to the invention (which is also optionally the solution subjected to ultrafiltration in step b) of the method according to the invention) is a solution further containing a buffer. In a specific embodiment, the solution provided in step a) of the invention is a composition containing an anti-CD20 antibody or a fragment thereof (especially a veltuzumab antibody) and a buffer. In a specific embodiment, the solution provided in step a) of the invention is a solution containing an IgG antibody or a fragment thereof and a buffer.

[0077] As used herein, buffer refers to a weak acid or base that allows the pH value of a solution to be maintained at an almost constant level, such as phosphates, acetates (e.g., sodium acetate), succinates (e.g., sodium succinate), gluconates, glutamates, citrates, other organic acid buffers, buffers containing amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine), in particular buffers containing histidine. Buffers containing amino acids are known to those skilled in the art and are selected according to their desired buffering properties. Exemplary buffers containing histidine are histidine, histidine chloride, histidine hydrochloride monohydrate, histidine acetate, histidine phosphate, histidine sulfate, and mixtures thereof. In particular, the buffer is histidine and / or histidine hydrochloride monohydrate. Optionally, the buffer is histidine. It is to be understood that any detailed description herein referring to histidine also relates to exemplary buffers, in particular the histidine buffers mentioned above. Further definitions of buffers comprising histidine are provided below.

[0078] In one embodiment of the present invention, the concentration of histidine in the solution provided in step a) of the method according to the present invention is 40 mM. In another embodiment of the present invention, the solution provided in step a) of the present invention has a pH value of 5.45. In particular, the solution provided in step a) of the method according to the present invention has a pH value of 5.45 and a histidine concentration of 40 mM.

[0079] The solution containing the antibody or fragment thereof may be provided by any method known to those skilled in the art. In one embodiment, a drug substance as a whole comprising an antibody or fragment thereof as defined herein is used as a starting material to provide a composition containing the antibody and, for example, histidine. Drug substances as a whole include, for example, those provided by Immunomedics and Boehringer Ingelheim, both of which contain the anti-CD 20 antibody veltuzumab. The starting material may then be purified to remove undesirable components and provide a starting concentration C S The invention relates to an antibody or fragment thereof and an optional buffer, such as L-histidine. Such undesirable ingredients may include, for example, sucrose, which may result in a rapid increase in the viscosity of the composition during the subsequent ultrafiltration step. High viscosity may reduce the antibody concentration obtained in the final product and generally hinder various method steps such as filtration. In the final product, high viscosity may have a negative impact on administration (e.g., syringe capacity).

[0080] Can be purified by any method known in the art such as filtering and centrifugation.Filtering includes any conventional filtration method for static filtration (such as vacuum filtration) and dynamic filtration (such as tangential flow filtration, diafiltration).In one embodiment of the method according to the present invention, purify by diafiltration (DF) or dialysis.In a specific embodiment, purify by diafiltration.In another specific embodiment, purify by dialysis.

[0081] "Dialysis" as used herein relates to any method in which molecules are purified by their different diffusion rates across a semipermeable membrane. Suitable methods of purification by dialysis are known to those skilled in the art.

[0082] As used herein, "diafiltration" refers to a process for removing solvents, replacing solvents or reducing solvent concentrations from compositions comprising proteins, peptides, nucleic acids or other biomolecules (particularly antibodies) using ultrafiltration membranes. Diafiltration provides the advantage that purification, concentration and, if necessary, buffer exchange can be performed in a single operating unit. Diafiltration devices are known in the art and include, for example, the tangential flow filtration (TFF) Unit Minim II provided by Pall.

[0083] Thus, in one embodiment of the invention, the method comprises the step of diafiltration of the solution of step a) with a buffer solution between step a) and step b), wherein the buffer is a buffer optionally comprising an amino acid, optionally histidine, in particular L-histidine. Thus, during the diafiltration of the starting material, a buffer exchange may be performed. It should be noted that any definition of a buffer comprising histidine given herein also relates to a buffer comprising an amino acid.

[0084] Exemplary buffers comprising histidine include histidine, in particular L-histidine, histidine chloride, histidine hydrochloride monohydrate, histidine acetate, histidine phosphate, histidine sulfate and mixtures thereof. In particular, a buffer comprising histidine is a buffer comprising histidine and / or histidine hydrochloride monohydrate. It should be understood that any detailed description of histidine mentioned herein also relates to the exemplary histidine buffers mentioned above. As used herein, a "buffer comprising histidine" relates to a pharmaceutically acceptable buffer comprising the amino acid histidine that is suitable for maintaining pH at an almost constant value.

[0085] In particular, the buffer is a buffer substance suitable for providing a pH value of 5.45 ± 1.0. More particularly, the buffer substance is a buffer substance suitable for providing a pH value of 5.45 ± 0.5. In particular, the buffer substance is a buffer substance suitable for providing a pH value of 5.45 ± 0.1. More particularly, the buffer substance is a buffer substance suitable for providing a pH value of 5.45 ± 0.05.

[0086] In one embodiment of the invention, the buffer solution comprising histidine as a buffer comprises 30-60 mM histidine, especially 35-45 mM histidine. In another embodiment of the invention, the buffer solution comprising histidine as a buffer comprises 30 mM histidine, 35 mM histidine, 40 mM histidine, 45 mM histidine, 50 mM histidine, 55 mM histidine or 60 mM histidine, especially a buffer solution comprising 40 mM histidine.

[0087] In a specific embodiment of the present invention, the buffer solution for diafiltration is a buffer solution comprising 30-60mM histidine, especially 35-45mM histidine and having a pH value of 5.45±2.0. In another specific embodiment of the present invention, the buffer solution for diafiltration is a buffer solution comprising 30-60mM histidine, especially 35-45mM histidine and having a pH value of 5.45±1.0. In another specific embodiment of the present invention, the buffer solution for diafiltration is a buffer solution comprising 30-60mM histidine, especially 35-45mM histidine and having a pH value of 5.45±0.5. In another specific embodiment of the present invention, the buffer solution for diafiltration is a buffer solution comprising 30-60mM histidine, especially 35-45mM histidine and having a pH value of 5.45±0.1. In another specific embodiment of the present invention, the buffer solution used for diafiltration is a buffer solution comprising 30-60 mM histidine, especially 35-45 mM histidine and having a pH value of 5.45±0.05.

[0088] In a specific embodiment of the present invention, the buffer solution used for diafiltration is a buffer solution comprising 40mM histidine and having a pH value of 5.45±2.0. In another specific embodiment of the present invention, the buffer solution used for diafiltration is a buffer solution comprising 40mM histidine and having a pH value of 5.45±1.0. In another specific embodiment of the present invention, the buffer solution used for diafiltration is a buffer solution comprising 40mM histidine and having a pH value of 5.45±0.5. In another specific embodiment of the present invention, the buffer solution used for diafiltration is a buffer solution comprising 40mM histidine and having a pH value of 5.45±0.1. In another specific embodiment of the present invention, the buffer solution used for diafiltration is a buffer solution comprising 40mM histidine and having a pH value of 5.45±0.05.

[0089] If buffer exchange is performed, it can be performed with at least 1 volume of buffer solution compared to the antibody solution, with at least 2 volumes of buffer solution compared to the antibody solution, with at least 3 volumes of buffer solution compared to the antibody solution, with at least 4 volumes of buffer solution compared to the antibody solution, with at least 5 volumes of buffer solution compared to the antibody solution, with at least 6 volumes of buffer solution compared to the antibody solution, with at least 7 volumes of buffer solution compared to the antibody solution, or with at least 8 volumes of buffer solution compared to the antibody solution. In particular, the buffer exchange is performed with at least 8 volumes of buffer solution compared to the antibody solution.

[0090] In step b) of the method for preparing HCLF according to the present invention, the antibody or fragment thereof is concentrated via ultrafiltration (UF) to obtain an antibody or fragment thereof having an intermediate concentration C I A solution of which C I In one embodiment of the invention, the solution subjected to ultrafiltration in step b) contains about 40 mM histidine and / or has a pH value of 5.45 ± 1.0. In another embodiment of the invention, the solution subjected to ultrafiltration in step b) contains about 40 mM histidine and / or has a pH value of 5.45 ± 0.5. In another embodiment of the invention, the solution subjected to ultrafiltration in step b) contains about 40 mM histidine and / or has a pH value of 5.45 ± 0.1.

[0091] In another embodiment, the solution subjected to ultrafiltration in step b) contains substantially no or no tonicity modifier as defined herein. In particular, the solution subjected to ultrafiltration in step b) contains substantially no or no sucrose.

[0092] In another embodiment, the solution subjected to ultrafiltration in step b) is substantially free of or free of a surfactant as defined herein. In particular, the solution subjected to ultrafiltration in step b) is substantially free of or free of a nonionic surfactant. More particularly, the solution subjected to ultrafiltration in step b) is substantially free of or free of polysorbate, wherein the polysorbate is optionally polysorbate 20.

[0093] Thus, according to one embodiment of the invention, the solution containing the antibody (optionally an anti-CD 20 antibody, in particular veltuzumab) and the buffer comprising an amino acid (optionally histidine) is substantially free of or free of tonicity modifiers and surfactants. In a specific embodiment, the solution subjected to ultrafiltration in step b) is an aqueous solution (i.e. a solution wherein the solvent is water) consisting of histidine and the antibody (as defined herein).

[0094] As used herein, "ultrafiltration" may refer to any membrane filtration method for purifying and / or concentrating macromolecular solutions, in which hydrostatic pressure is used to filter a liquid through a semipermeable membrane having appropriate chemical and physical properties. Such ultrafiltration membranes are membranes having a pore size of 0.01-0.1 μm and are used to separate particles having a size of about 0.1-0.01 μm from a suspension, such as emulsions, proteins, and macromolecules. Antibodies as described herein are concentrated to as much as above C. H and an intermediate concentration C of at least about 260 mg / mL I Usually, C I In the range of about 260-290 mg / mL, in particular about 270-290 mg / mL, more particularly about 270-285 mg / mL. In one embodiment of the invention, the antibody as described herein is concentrated to a concentration of up to at least 260 mg / mL. I In some embodiments of the invention, the antibodies described herein are concentrated to a concentration of at least 265 mg / mL, at least 270 mg / mL, at least 275 mg / mL, at least 280 mg / mL, at least 285 mg / mL, at least 290 mg / mL. I , especially up to a concentration of at least 285 mg / mL C I As discussed above the term "about 280 mg / mL" relates to minor variations within ±10%, optionally ±5% of the numerical value (ie 280 mg / mL). In one embodiment, the antibody is concentrated to an intermediate concentration of up to 280 mg / mL.

[0095] In one embodiment of the method for preparing the HCLF of an antibody or its fragment, step b) is carried out in an ultrafiltration device and the method further comprises the step of rinsing the device for ultrafiltration with a buffer solution (optionally a buffer solution comprising amino acids as defined herein) between step b) and step c). In particular, the buffering agent in the buffer solution for rinsing the ultrafiltration device is histidine, particularly L-histidine. Additionally or alternatively, the buffering agent used for rinsing is the same as the buffering agent contained in the solution of step a). This rinsing step allows further minimization of antibody losses when removing the highly concentrated antibody composition from the ultrafiltration device, thereby further maximizing the yield of the method described herein.

[0096] In a further step c) of the method described herein, the highly concentrated antibody composition obtained in step b) may be further diluted to obtain a desired final concentration of HCLF.

[0097] In step c), the desired final concentration of the antibody or fragment thereof in HCLF may be obtained by any method known in the art. The method selected and the components used to obtain the desired final concentration of the antibody or fragment thereof may depend on the desired final composition of the HCLF.

[0098] In one embodiment thereof, step c) of the method according to the invention thus comprises diluting the highly concentrated antibody composition obtained in step c) with a buffer solution.

[0099] The buffer solution may comprise any of the aforementioned buffers, such as a buffer comprising amino acids, in particular a buffer comprising histidine. In particular, the buffer solution comprises 30-50 mM histidine, optionally 30 mM histidine.

[0100] In addition, the pH value of the buffer solution used to dilute the highly concentrated antibody composition obtained from step b) can be in the range of 4.8 to 7.0, especially in the range of 5 to 6. Optionally, the pH value of the buffer solution is 5.5±2.0, 5.5±1.0, 5.5±0.5, 5.5±0.3 or 5.5±0.2. In one embodiment, the pH value of the buffer solution is 5.5±0.3. In another embodiment, the pH value of the buffer solution is 5.5±0.2.

[0101] In particular, the buffer solution may be a buffer solution further comprising a surfactant as defined herein. In one embodiment of the method according to the invention, the buffer solution comprising a surfactant is a buffer solution comprising a nonionic surfactant, especially a polysorbate. In a specific embodiment of the method according to the invention, the buffer solution comprising a surfactant is a buffer solution comprising polysorbate 20 or polysorbate 80, especially polysorbate 20.

[0102] The buffer solution may contain 5 times, 6 times, 7 times, 8 times, 9 times or 10 times the concentration of the desired surfactant concentration in the final product. In a specific embodiment, the buffer solution has a concentration of 10 times the concentration of the desired surfactant concentration in the final product.

[0103] In particular, the buffer solution comprises a surfactant concentration of at least 0.1 g / L, at least 0.2 g / L, at least 0.3 g / L, at least 0.4 g / L, at least 0.5 g / L, at least 0.6 g / L, at least 0.7 g / L, at least 0.8 g / L, at least 0.9 g / L, at least 1 g / L, at least 2 g / L, at least 3 g / L, at least 4 g / L, at least 5 g / L, at least 6 g / L, at least 7 g / L, at least 8 g / L, at least 9 g / L or at least 10 g / L.

[0104] In one embodiment, the concentration of the surfactant in the buffer solution is in the range of 0.1-1 g / L, especially in the range of 0.3 g / L to 1 g / L. In this embodiment, the surfactant is especially polysorbate 80. In another embodiment, the concentration of the surfactant in the buffer solution is in the range of 1-10 g / L, especially in the range of 1-3 g / L. In this embodiment, the surfactant is especially polysorbate 20.

[0105] In a specific embodiment, the buffer solution comprises 0.2 g / L surfactant, especially 0.2 g / L polysorbate 80. In another specific embodiment, the buffer solution comprises 2 g / L surfactant, especially 2 g / L polysorbate 20.

[0106] The buffer solution may further comprise a tension modifier. The tension modifier may be selected from NaCl, potassium chloride, glycine, glycerol, salts, amino acids and sugars. Optionally, the tension modifier is a sugar or a sugar alcohol, for example selected from sucrose, trehalose, mannitol, sorbitol and glucose. In particular, the tension modifier is selected from sorbitol, sucrose and / or mannitol. In one embodiment, the tension modifier is sorbitol. In one embodiment, the tension modifier is mannitol. In one embodiment, the tension modifier is sucrose.

[0107] The buffer solution may contain 5 times, 6 times, 7 times, 8 times, 9 times or 10 times the desired concentration of the tonicity modifier in the final product. In particular, the buffer solution contains a tonicity modifier concentration that is 10 times the desired concentration of the tonicity modifier in the final product.

[0108] In particular, the buffer solution may comprise a tonicity modifier of at least 1 M, at least 1.5 M, at least 2 M, at least 2.2 M, at least 2.4 M, at least 2.6 M, at least 2.8 M, at least 3 M. Optionally, the concentration of the tonicity modifier in the buffer solution of the present invention is in the range of 0.05 M to 5.5 M, in particular 1 to 3 M, more in particular 2 to 2.5 M. In a specific embodiment of the present invention, the concentration of the tonicity modifier is 2.2 M.

[0109] The solution derived from the dilution step c) may have a concentration of at least 50 mg / mL, at least 70 mg / mL, at least 75 mg / mL, at least 80 mg / mL, at least 90 mg / mL, at least 100 mg / mL, at least 110 mg / mL, at least 120 mg / mL, at least 130 mg / mL, at least 140 mg / mL, at least 150 mg / mL, at least 155 mg / mL, at least 160 mg / mL, at least 170 mg / ml, at least 175 mg / mL, at least 180 mg / mL, at least 190 mg / mL or at least 200 mg / mL, in particular at least 175 mg / mL of the antibody or fragment thereof. In one embodiment, the composition obtained in the dilution step has a concentration of 100-290 mg / mL, 130-200 mg / mL, 155-200 mg / mL, 160-200 mg / mL or 175-200 mg / mL of the antibody or fragment thereof.

[0110] The concentration of surfactant in the composition derived from the dilution step can be any concentration that a person skilled in the art considers suitable. In particular, in the antibody composition after dilution, the composition may include at least 0.01 g / L, at least 0.02 g / L, at least 0.03 g / L, at least 0.04 g / L, at least 0.05 g / L, at least 0.06 g / L, at least 0.07 g / L, at least 0.08 g / L, at least 0.09 g / L, at least 0.1 g / L, at least 0.2 g / L, at least 0.3 g / L, at least 0.4 g / L, at least 0.5 g / L, at least 0.6 g / L, at least 0.7 g / L, at least 0.8 g / L, at least 0.9 g / L or at least 1.0 g / L surfactant concentration. In one embodiment, the concentration of surfactant in the composition after dilution is in the range of 0.01-0.1 g / L, especially 0.03 g / L to 0.1 g / L. In this embodiment, the surfactant is especially polysorbate 80.

[0111] In another embodiment, the concentration of the surfactant in the composition after dilution is in the range of 0.1-1.0 g / L, especially 0.1-0.3 g / L. In this embodiment, the surfactant is especially polysorbate 20.

[0112] In one embodiment, the concentration of the surfactant in the composition after dilution is 0.02 g / L surfactant, especially 0.02 g / L polysorbate 80. In another embodiment, the concentration of the surfactant in the composition after dilution is 0.2 g / L surfactant, especially 0.2 g / L polysorbate 20.

[0113] The concentration of the tension modifier in the composition resulting from the dilution step may be any concentration deemed suitable by a person skilled in the art. In particular, the composition may comprise a tension modifier concentration of at least 100 mM, at least 150 mM, at least 200 mM, at least 220 mM, at least 240 mM, at least 260 mM, at least 280 mM, at least 300 mM. Optionally, the concentration of the tension modifier in the antibody composition after dilution is in the range of 100-300 mM, in particular 200-250 mM.

[0114] Step c) of the method according to the invention may comprise a second dilution step for final adjustment of the concentration of the antibody or fragment thereof in the HCLF. In particular, this step comprises dilution with a so-called formulation buffer solution.

[0115] The formulation buffer solution used in step c) may contain a surfactant as defined herein. In one embodiment, the surfactant is a nonionic surfactant, especially a polysorbate. In a specific embodiment, the surfactant is polysorbate 20 or polysorbate 80, especially polysorbate 20. The concentration of the surfactant in the HCLF obtained according to the method of the present invention is at least 0.01 g / L, at least 0.02 g / L, at least 0.03 g / L, at least 0.04 g / L, at least 0.05 g / L, at least 0.06 g / L, at least 0.07 g / L, at least 0.08 g / L, at least 0.09 g / L, at least 0.1 g / L, at least 0.2 g / L, at least 0.3 g / L, at least 0.4 g / L, at least 0.5 g / L, at least 0.6 g / L, at least 0.7 g / L, at least 0.8 g / L, at least 0.9 g / L or at least 1.0 g / L surfactant concentration.

[0116] In one embodiment, the concentration of the surfactant in the formulation buffer solution of the present invention is in the range of 0.01-0.1 g / L, especially 0.03 g / L to 0.1 g / L. In this embodiment, the surfactant is especially polysorbate 80.

[0117] In another embodiment, the concentration of the surfactant in the formulation buffer solution is in the range of 0.1-1.0 g / L, especially 0.1-0.3 g / L. In this embodiment, the surfactant is especially polysorbate 20.

[0118] The formulation buffer solution used in the method of the present invention may further comprise a tension modifier. The tension modifier may be selected from NaCl, potassium chloride, glycine, glycerol, salt, amino acid, sugar alcohol and sugar. Optionally, the tension modifier is a sugar or sugar alcohol, for example selected from glucose, sucrose, trehalose, mannitol, sorbitol and glucose. In particular, the tension modifier is sorbitol and / or mannitol. In one embodiment, the tension modifier is sorbitol. The concentration of the tension modifier in the buffer solution may be any concentration deemed suitable by a person skilled in the art. In particular, the formulation buffer solution used in the method of the present invention may comprise at least 100mM, at least 150mM, at least 200mM, at least 220mM, at least 240mM, at least 260mM, at least 280mM, at least 300mM tension modifier. Optionally, the concentration of the tension modifier in the formulation buffer solution used in the method of the present invention is in the range of 100-300mM, especially 200-250mM. In a specific embodiment, the formulation buffer solution comprises 220 mM tonicity modifier, in particular 220 mM sorbitol.

[0119] The formulation buffer solution further comprises a buffer as defined herein. In particular, the buffer is a buffer comprising an amino acid, optionally histidine, in particular L-histidine, histidine chloride, histidine hydrochloride monohydrate, histidine acetate, histidine phosphate or histidine sulfate or a mixture thereof. In particular, the formulation buffer solution comprises histidine and / or histidine hydrochloride monohydrate.

[0120] In one embodiment of the invention, the formulation buffer solution comprises 30-60 mM histidine, especially 35-45 mM histidine. In another embodiment of the invention, the formulation buffer solution comprises about 30 mM histidine, about 35 mM histidine, about 40 mM histidine, about 45 mM histidine, about 50 mM histidine, about 55 mM histidine or about 60 mM histidine, especially the formulation buffer solution comprises 30 mM histidine. In another embodiment, the formulation buffer solution comprises at least 50 mM histidine.

[0121] In addition, the pH value of the formulation buffer solution used in step c) may be in the range of 4.8 to 7.0, especially in the range of 5 to 6. Optionally, the pH value of the buffer solution is 5.5 ± 2.0, 5.5 ± 1.0, 5.5 ± 0.5, 5.5 ± 0.3 or 5.5 ± 0.2. In one embodiment, the pH value of the buffer solution is 5.5 ± 0.3. In another embodiment, the pH value of the buffer solution is 5.5 ± 0.2.

[0122] The methods as described herein are performed at room temperature, as defined above. In particular, all method steps as described herein are performed at 18°C ​​to 25°C, optionally at 19°C to 22°C.

[0123] The HCLF obtained according to the method of the present invention has a concentration of antibodies or fragments thereof as defined above for HCLF.

[0124] The pH value of the HCLF obtained according to the method of the present invention may be in the range of 4.8 to 7.0, in particular in the range of 5.0 to 6.0. In one embodiment, the HCLF obtained according to the method of the present invention has a pH of 5.5 ± 2.0, 5.5 ± 1.0, 5.5 ± 0.5, 5.5 ± 0.3 or 5.5 ± 0.2. In a specific embodiment, the pH value of the liquid pharmaceutical preparation of the present invention is 5.5 ± 0.3. In another specific embodiment, the pH value of the liquid pharmaceutical preparation of the present invention is 5.5 ± 0.2.

[0125] The HCLF obtained according to the process of the present invention comprises a buffer as defined herein. In particular, the buffer is an amino acid-containing buffer, optionally comprising histidine, in particular L-histidine, histidine chloride, histidine hydrochloride monohydrate, histidine acetate, histidine phosphate or histidine sulfate or a mixture thereof. In particular, the HCLF obtained according to the process of the present invention comprises histidine and / or histidine hydrochloride monohydrate.

[0126] In one embodiment of the present invention, the HCLF obtained according to the method of the present invention comprises 30-60 mM histidine, especially 35-45 mM histidine. In another embodiment of the present invention, the HCLF obtained according to the method of the present invention comprises about 30 mM histidine, about 35 mM histidine, about 40 mM histidine, about 45 mM histidine, about 50 mM histidine, about 55 mM histidine or about 60 mM histidine, especially the HCLF obtained according to the method of the present invention comprises 30 mM histidine. In another embodiment, the HCLF comprises at least 50 mM histidine.

[0127] The HCLF obtained according to the method of the present invention may contain a surfactant as defined herein. In one embodiment, the surfactant is a nonionic surfactant, especially a polysorbate. In a specific embodiment, the surfactant is polysorbate 20 or polysorbate 80, especially polysorbate 20. The concentration of the surfactant in the HCLF obtained according to the method of the present invention is at least 0.01 g / L, at least 0.02 g / L, at least 0.03 g / L, at least 0.04 g / L, at least 0.05 g / L, at least 0.06 g / L, at least 0.07 g / L, at least 0.08 g / L, at least 0.09 g / L, at least 0.1 g / L, at least 0.2 g / L, at least 0.3 g / L, at least 0.4 g / L, at least 0.5 g / L, at least 0.6 g / L, at least 0.7 g / L, at least 0.8 g / L, at least 0.9 g / L or at least 1.0 g / L surfactant concentration.

[0128] In one embodiment, the concentration of the surfactant in the HCLF obtained according to the method of the present invention is in the range of 0.01-0.1 g / L, especially 0.03 g / L to 0.1 g / L. In this embodiment, the surfactant is especially polysorbate 80.

[0129] In another embodiment, the concentration of the surfactant in the diluted composition is in the range of 0.1-1.0 g / L, especially 0.1-0.3 g / L. In this embodiment, the surfactant is especially polysorbate 20.

[0130] The HCLF obtained according to the inventive method may further comprise a tension modifier. The tension modifier may be selected from NaCl, potassium chloride, glycine, glycerol, salt, amino acid, sugar alcohol and sugar. Optionally, the tension modifier is a sugar or sugar alcohol, for example selected from glucose, sucrose, trehalose, mannitol, sorbitol and glucose. In particular, the tension modifier is sorbitol and / or mannitol. In one embodiment, the tension modifier is sorbitol. The concentration of the tension modifier in the buffer may be any concentration deemed suitable by a person skilled in the art. In particular, the HCLF obtained according to the inventive method may comprise at least 100 mM, at least 150 mM, at least 200 mM, at least 220 mM, at least 240 mM, at least 260 mM, at least 280 mM, at least 300 mM tension modifier. Optionally, the concentration of the tension modifier in the HCLF obtained according to the method of the present invention is in the range of 5.5 mM-550 mM, in particular 100-300 mM, more particularly 200-250 mM. In a specific embodiment, the concentration of the tension modifier in the HCLF obtained according to the method of the present invention is 220 mM. In another embodiment of the present invention, the concentration of the tension modifier of the present invention is at least 270 mM.

[0131] It should be understood that the HCLF obtained according to the inventive method may further comprise any other pharmaceutically acceptable compounds that are considered useful by those skilled in the art. Examples of other pharmaceutically acceptable compounds include pharmaceutically acceptable excipients, additives, diluents, chelating agents, freeze-drying protectants, adjuvants, delivery vehicles and antimicrobial preservatives, which may be added to provide the desired properties for the final formulation. Such additional compounds are known to those skilled in the art and may be selected according to the desired properties. They may be added at any step during the above-mentioned method that is considered suitable by those skilled in the art. In particular, other pharmaceutically acceptable compounds may be added during the first dilution step of step c).

[0132] In one embodiment, the HCLF obtained according to the method of the present invention further comprises arginine at a concentration deemed suitable by a person skilled in the art. Adding arginine allows the antibody to be further stabilized and the melting point to be increased.

[0133] An exemplary embodiment of the method for providing HCLF according to the present invention is shown in Figures 1 to 3 and described in detail in the accompanying drawings.

[0134] During storage, the antibody preparation may undergo chemical and / or physical degradation, resulting in unwanted degradation products such as aggregates. This degradation may result in reduced effectiveness of the antibody and increased risk of unwanted side effects (such as unwanted immune responses), which may result in a significant impact on regulatory approval of the drug. In particular, at higher concentrations of antibodies, the tendency to form aggregates may be significantly increased. However, it has been found that by using an aqueous solution comprising amino acids, anti-CD 20 antibodies or fragments thereof can be stabilized and dissolved in liquid pharmaceutical preparations at high concentrations. Amino acids include any amino acids that are considered suitable by those skilled in the art, such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. In particular, the amino acid should serve as a buffer. In one embodiment of the invention, the aqueous solution comprises histidine, optionally L-histidine. It should be noted that any detailed description provided herein for histidine is also applicable to L-histidine.

[0135] In another aspect, the present invention therefore relates to a method for stabilizing an anti-CD 20 antibody or fragment thereof at a concentration of at least 155 mg / mL in a liquid pharmaceutical formulation by mixing said antibody or fragment thereof (which has not been previously subjected to lyophilization or freeze drying) with an aqueous solution comprising an amino acid, optionally histidine.

[0136] In one embodiment thereof, the liquid pharmaceutical preparation comprises at least 160 mg / mL, at least 175 mg / mL, at least 180 mg / mL, at least 185 mg / mL, at least 190 mg / mL, at least 195 mg / mL, at least 200 mg / mL, at least 220 mg / mL, at least 240 mg / mL, at least 260 mg / mL, at least 280 mg / mL, at least 285 mg / mL or at least 290 mg / mL of veltuzumab antibody or its fragment. In a specific embodiment, the liquid pharmaceutical preparation comprises at least 160 mg / mL of veltuzumab antibody or its fragment. In another specific embodiment, the liquid pharmaceutical preparation comprises at least 175 mg / mL of veltuzumab antibody or its fragment. In another specific embodiment, the liquid pharmaceutical preparation comprises at least 200 mg / mL of veltuzumab antibody or its fragment. In another embodiment of the present invention, the liquid pharmaceutical formulation of the present invention comprises 160-290 mg / mL, 160-250 mg / mL, 160-220 mg / mL, 160-200 mg / mL or 160-175 mg / mL of the veltuzumab antibody or a fragment thereof.

[0137] According to the method of the present invention, the antibody or fragment thereof is not previously subjected to lyophilization or freeze drying. "Lyophilization" as used herein refers to any dehydration method in which the preparation is frozen and the ambient pressure is subsequently reduced to allow the frozen water to sublime from the frozen preparation. Such methods can be performed by any lyophilization method known in the art.

[0138] In the method according to the invention, the antibody or fragment thereof is mixed with an aqueous solution comprising amino acids, optionally histidine. As used herein, an aqueous solution relates to any solution in which the solvent is water.

[0139] The aqueous solution comprising histidine may comprise only histidine as a buffer, histidine chloride, histidine hydrochloride monohydrate, histidine acetate, histidine phosphate or histidine sulfate or a combination thereof. In particular, the aqueous solution comprising histidine used in the method of the present invention comprises only histidine as a buffer or a combination of histidine and histidine hydrochloride monohydrate as a buffer.

[0140] In addition to the amino acid, optionally in addition to the above-mentioned histidine or in addition to the above-mentioned combination of buffers containing histidine, the aqueous solution comprising amino acids (optionally histidine) may comprise at least one other therapeutically acceptable buffer. Such other buffers include phosphates, acetates (e.g., sodium acetate), succinates (e.g., sodium succinate), gluconates, glutamate, histidine, citrates, other amino acids mentioned herein that act as buffers, and other organic acid buffers.

[0141] The pH value of the aqueous solution comprising amino acids (optionally histidine) can be in the range of 4.8 to 7.0, especially in the range of 5.0 to 6.0. In one embodiment, the pH value of the aqueous solution comprising amino acids (optionally histidine) can be 5.5 ± 2.0, 5.5 ± 1.0, 5.5 ± 0.5, 5.5 ± 0.3 or 5.5 ± 0.2. In a specific embodiment, the pH value of the aqueous solution comprising amino acids (optionally histidine) used in the method of the present invention is 5.5 ± 0.3. In another specific embodiment, the pH value of the aqueous solution comprising amino acids (optionally histidine) used in the method of the present invention is 5.5 ± 0.2.

[0142] In one embodiment of the invention, the aqueous solution comprising amino acid (optionally histidine) used in the inventive method comprises 30-60mM amino acid, optional histidine, especially 30-45mM amino acid, optional histidine. In another embodiment of the invention, the aqueous solution comprising amino acid (optionally histidine) used in the inventive method comprises 30mM amino acid, optional histidine, 35mM amino acid, optional histidine, 40mM amino acid, optional histidine, 45mM amino acid, optional histidine, 50mM amino acid, optional histidine, 55mM amino acid, optional histidine or 60mM amino acid, optional histidine. In particular, the aqueous solution comprising amino acid (optionally histidine) used in the inventive method comprises 30mM amino acid, optional histidine. In another embodiment, the aqueous solution comprising amino acid (optionally histidine) comprises at least 50mM amino acid, optional histidine.

[0143] The aqueous solution containing amino acids (optionally histidine) used in the method of the present invention may further contain a tension modifier. The tension modifier may be selected from NaCl, potassium chloride, glycine, glycerol, salts, amino acids, sugar alcohols and sugars. Optionally, the tension modifier is a sugar or a sugar alcohol, for example selected from glucose, sucrose, trehalose, mannitol, sorbitol and glucose. In particular, the tension modifier is sorbitol, sucrose and / or mannitol. In one embodiment, the tension modifier is sorbitol. In another embodiment, the tension modifier is sucrose. In another embodiment, the tension modifier is mannitol. The concentration of the tension modifier in the buffer may be any concentration deemed suitable by a person skilled in the art. The aqueous solution containing amino acids (optionally histidine) used in the method of the present invention may contain at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9% or at least 10% tension modifier. In one embodiment of the present invention, the aqueous solution containing amino acids (optionally histidine) contains 5-8% tension modifier.

[0144] The addition of a surfactant leads to a further reduction in the formation of degradation products, especially a reduction in the formation of aggregates, thereby contributing to the stabilization of the antibody formulation. Therefore, the aqueous solution comprising amino acids (optionally histidine) used in the method of the present invention may comprise a surfactant as defined herein. In one embodiment, the surfactant is a nonionic surfactant, especially a polysorbate. In a specific embodiment, the surfactant is polysorbate 20 or polysorbate 80, especially polysorbate 20. The aqueous solution comprising amino acids (optionally histidine) used in the method according to the present invention may contain any amount of surfactant that a person skilled in the art considers useful, especially at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.1% or at least 1.0% surfactant. Optionally, the aqueous solution comprising amino acids (optionally histidine) used in the method according to the present invention contains at least 0.01% surfactant.

[0145] Examples of anti-CD20 antibodies that can be stabilized according to the above method are veltuzumab, rituximab, ocrelizumab, ofatumumab, 90 Ibritumomab tiuxetan 131 Tositumomab, TRU-015, AME-133v, humanized PRO131921, GA101, 1F5 IgG2a, HI47 IgG3, 2C6 IgG1, 2H7 IgG1, AT80 IgG1, 11B8IgG1, humanized B-Ly1 antibody IgG1, and Aftuzumab (HuMab <cd20>) or a fragment thereof. In particular, the anti-CD20 antibody stabilized by the above method is veltuzumab or a fragment thereof.

[0146] As used herein, "stable" or "stabilized" in relation to the antibody formulations of the invention relates to antibody formulations that maintain their chemical stability and / or physical stability during storage. Methods for determining the stability of antibody formulations are known in the art. In particular, stable / stabilized antibody formulations maintain their chemical and / or physical stability for at least one month when stored at room temperature, optionally for at least three months when stored at 25° C. and / or for at least one year when stored at 2-8° C.

[0147] In a specific embodiment, the liquid formulation of the anti-CD20 antibody or fragment thereof provided by any of the above methods comprises a) at least 160 mg / mL of an anti-CD20 antibody or fragment thereof, optionally veltuzumab antibody or fragment thereof; b) 220 mM sorbitol; c) 30 mM histidine; d) 0.2 g / L polysorbate 20; and And have a pH value in the range of 4.8 to 7.0, especially in the range of 5.0 to 6.0. In a specific embodiment, the above-mentioned liquid preparation has a pH value of 5.5±0.3. In another specific embodiment, the above-mentioned liquid preparation has a pH value of 5.5±0.2.

[0148] The liquid formulation of the anti-CD20 antibody or fragment thereof provided by the above method is suitable for long-term storage and can also be lyophilized in order to even increase the shelf life.

[0149] Exemplary methods for stabilizing anti-CD 20 antibodies are shown in the Examples below.

[0150] It has further been found that a liquid pharmaceutical formulation comprising at least 155 mg / mL of a veltuzumab antibody or fragment thereof can be provided with a reduced tendency to form aggregates.

[0151] In one aspect, the present invention therefore relates to a liquid pharmaceutical preparation of a veltuzumab antibody or its fragment, the liquid pharmaceutical preparation comprising at least 155mg / mL veltuzumab antibody or its fragment and amino acids.Amino acids include any amino acids that those skilled in the art consider suitable, such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. In particular, the amino acids should serve as buffers. In one embodiment of the invention, the liquid pharmaceutical preparation comprises histidine, optionally L-histidine. It should be noted that any detailed description provided herein for histidine is also applicable to L-histidine.

[0152] In one embodiment thereof, the liquid pharmaceutical preparation comprises at least 160 mg / mL, at least 175 mg / mL, at least 180 mg / mL, at least 185 mg / mL, at least 190 mg / mL, at least 195 mg / mL, at least 200 mg / mL, at least 220 mg / mL, at least 240 mg / mL, at least 260 mg / mL, at least 280 mg / mL, at least 285 mg / mL or at least 290 mg / mL of veltuzumab antibody or its fragment. In a specific embodiment, the liquid pharmaceutical preparation comprises at least 160 mg / mL of veltuzumab antibody or its fragment. In another specific embodiment, the liquid pharmaceutical preparation comprises at least 175 mg / mL of veltuzumab antibody or its fragment. In another specific embodiment, the liquid pharmaceutical preparation comprises at least 200 mg / mL of veltuzumab antibody or its fragment. In another embodiment of the present invention, the liquid pharmaceutical formulation of the present invention comprises 160-290 mg / mL, 160-250 mg / mL, 160-220 mg / mL, 160-200 mg / mL or 160-175 mg / mL of the veltuzumab antibody or a fragment thereof.

[0153] The liquid pharmaceutical formulation of the present invention comprises amino acids, optionally histidine as a buffer. It may comprise only amino acids, optionally histidine as a buffer or additionally histidine, histidine chloride, histidine hydrochloride monohydrate, histidine acetate, histidine phosphate or histidine sulfate or a combination thereof. In particular, the liquid pharmaceutical formulation of the present invention comprises only histidine as a buffer or a combination of histidine and histidine hydrochloride monohydrate as a buffer.

[0154] In addition to the amino acids mentioned above, optionally histidine or in addition to the combination of buffers containing histidine mentioned above, the liquid pharmaceutical formulation of the present invention may contain at least one other therapeutically acceptable buffer. Such other buffers include phosphates, acetates (e.g., sodium acetate), succinates (e.g., sodium succinate), gluconates, glutamate, histidine, citrates, other amino acids and other organic acid buffers.

[0155] In one embodiment of the invention, the concentration of the buffer, optionally histidine, in the liquid pharmaceutical formulation of the invention is in the range of 1 mM to 100 mM. In another embodiment, the concentration of the buffer, optionally histidine, in the liquid pharmaceutical formulation of the invention is in the range of 10 mM to 60 mM or 10 mM to 50 mM, optionally in the range of 20 mM to 40 mM. The liquid pharmaceutical formulation of the invention may comprise 10 mM of buffer, optionally histidine, 15 mM of buffer, optionally histidine, 20 mM of buffer, optionally histidine, 25 mM of buffer, optionally histidine, 30 mM of buffer, optionally histidine, 35 mM of buffer, optionally histidine, 40 mM of buffer, optionally histidine, 45 mM of buffer, optionally histidine, 50 mM of buffer, optionally histidine, 55 mM of buffer, optionally histidine or 60 mM of buffer, optionally histidine, in particular it comprises 30 mM of buffer, optionally histidine. In another embodiment, it comprises at least 50 mM of a buffer, optionally histidine.

[0156] The liquid pharmaceutical formulation of the present invention may further comprise a surfactant as defined herein. The addition of a surfactant may result in a further reduction in the formation of degradation products, especially in the formation of aggregates. In one embodiment, the surfactant is a nonionic surfactant, especially a polysorbate. In a specific embodiment, the surfactant is polysorbate 20 or polysorbate 80, especially polysorbate 20. The liquid pharmaceutical formulation according to the present invention may comprise any amount of surfactant that a person skilled in the art would consider useful. The liquid pharmaceutical formulation according to the present invention may comprise 0.01 to 1.0 g / L of a surfactant. In particular, it may contain at least 0.01 g / L, at least 0.02 g / L, at least 0.03 g / L, at least 0.04 g / L, at least 0.05 g / L, at least 0.06 g / L, at least 0.07 g / L, at least 0.08 g / L, at least 0.09 g / L, at least 0.01 g / L at least 0.02 g / L, at least 0.03 g / L, at least 0.04 g / L, at least 0.05 g / L, at least 0.1 g / L, at least 0.2 g / L or at least 1.0 g / L of surfactant.

[0157] In one embodiment, the concentration of the surfactant in the liquid pharmaceutical formulation of the present invention is in the range of 0.01-0.1 g / L, especially 0.03 g / L-0.1 g / L. In this embodiment, the surfactant is especially polysorbate 80.

[0158] In another embodiment, the concentration of the surfactant in the liquid pharmaceutical formulation is in the range of 0.1-1.0 g / L, especially 0.1-0.3 g / L. In this embodiment, the surfactant is especially polysorbate 20.

[0159] The liquid pharmaceutical preparation of the present invention may further comprise a tension modifier. The tension modifier may be selected from NaCl, potassium chloride, glycine, glycerol, salt, amino acid, sugar alcohol and sugar. Optionally, the tension modifier is a sugar or sugar alcohol, for example selected from glucose, sucrose, trehalose, mannitol, sorbitol and glucose. In particular, the tension modifier is sorbitol, sucrose and / or mannitol, more particularly sorbitol and / or mannitol. In one embodiment, the tension modifier is sorbitol. In another embodiment, the tension modifier is sucrose. In another embodiment, the tension modifier is mannitol. The concentration of the tension modifier in the buffer may be any concentration deemed suitable by a person skilled in the art. In one embodiment of the present invention, the concentration of the tension modifier in the liquid pharmaceutical preparation of the present invention is in the range of 5.5mM-550mM, in particular 100-300mM, more particularly 200-250mM. In a specific embodiment, the concentration of the tension modifier in the buffer is 220mM. In particular, the concentration of the tonicity modifier in the liquid pharmaceutical formulation of the present invention is at least 100 mM, at least 150 mM, at least 200 mM, at least 220 mM, at least 240 mM, at least 260 mM, at least 280 mM or at least 300 mM tonicity modifier. In one embodiment of the present invention, the concentration of the tonicity modifier of the present invention is at least 220 mM. In another embodiment of the present invention, the concentration of the tonicity modifier of the present invention is at least 270 mM.

[0160] The pH value of the liquid pharmaceutical preparation of the present invention can be in the range of 4.8 to 7.0, especially in the range of 5.0 to 6.0. In one embodiment of the present invention, the pH value of the liquid pharmaceutical preparation of the present invention is 5.5 ± 2.0, 5.5 ± 1.0, 5.5 ± 0.5, 5.5 ± 0.3 or 5.5 ± 0.2. In a specific embodiment, the pH value of the liquid pharmaceutical preparation of the present invention is 5.5 ± 0.3. In another specific embodiment, the pH value of the liquid pharmaceutical preparation of the present invention is 5.5 ± 0.2.

[0161] The pH of the liquid pharmaceutical preparation of the present invention can be adjusted with any acid or base deemed suitable by a person skilled in the art, especially a pharmaceutically acceptable acid or base. Examples include hydrochloric acid, sodium hydroxide and acetic acid.

[0162] In one embodiment of the present invention, the liquid pharmaceutical preparation according to the present invention comprises a) at least 160 mg / mL veltuzumab antibody or fragment thereof; b) 220 mM sorbitol; c) 30 mM histidine; d) 0.2 mg / mL polysorbate 20; and And have a pH value in the range of 4.8 to 7.0, especially in the range of 5.0 to 6.0. In a specific embodiment, the above liquid formulation has a pH value of 5.5±0.3. Optionally, the liquid pharmaceutical formulation has a pH value of 5.5±0.2.

[0163] The liquid pharmaceutical preparation of the present invention may further comprise additional components, such as pharmaceutically acceptable excipients, additives, diluents, chelating agents, lyophilization protectants, adjuvants, delivery vehicles and antimicrobial preservatives, which may be added to provide the desired properties for the final formulation. Such additional components are known to those skilled in the art and may be selected according to the desired properties. In one embodiment, the liquid pharmaceutical preparation of the present invention further comprises arginine, which is used to further stabilize and increase the melting point at concentrations deemed suitable by those skilled in the art.

[0164] Another embodiment of the present invention relates to a liquid pharmaceutical formulation as described herein for use as a medicament. It should also be understood herein that the HCLF derived by the above method or the liquid pharmaceutical formulation stabilized by the above method may also be used as a medicament and the description given below about the liquid pharmaceutical formulation also relates to any formulation derived by the above method.

[0165] In one embodiment, the liquid pharmaceutical formulation or HCLF as described herein can be used to treat cancer or a non-malignant disease, optionally an inflammatory or autoimmune disease.

[0166] As used herein, "cancer" may refer to any malignant disease involving unregulated cell growth, such as Epstein-Barr virus infection, leukemia, lymphoma, plasmacytoma, tumor virus infection, immunoproliferative disorder, lymphoproliferative disorder, atypical proteinemia, herpes virus infection, DNA virus infection.

[0167] In particular, the liquid pharmaceutical formulation or HCLF as described herein can be used to treat any CD20 positive cancer, i.e. a cancer showing abnormal proliferation of cells which express CD20 on the cell surface, in particular T-cells or B-cells. Methods for determining CD20 expression on the cell surface are known in the art and include, for example, FACS or PCR. Exemplary CD20 positive cancers that can be treated with the liquid pharmaceutical formulation or HCLF according to the invention are B cell lymphomas and leukemias.

[0168] Lymphomas and leukemias include Burkitt's lymphoma, B-cell leukemia, chronic lymphocytic B-cell leukemia, acute lymphocytic leukemia, lymphoid leukemia, prolymphocytic leukemia, hairy cell leukemia, multiple myeloma, post-transplant lymphoproliferative disorder (PTLD), HIV-associated lymphoma, primary CNS lymphoma, B-cell lymphoma, marginal zone B-cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, immunoblastic large cell lymphoma, mantle cell lymphoma, non-Hodgkin's lymphoma (e.g., follicular lymphoma), lymphomatoid granuloma, precursor cell lymphocytic leukemia-lymphoma, Waldenstrom's macroglobulinemia, prolymphocytic lymphoma, diffuse large B-cell lymphoma, immunoblastic large cell lymphoma, mantle cell lymphoma, lymphomatoid granuloma, and precursor cell lymphocytic leukemia-lymphoma. Optionally, the CD 20-positive cancer is a disease selected from the group consisting of B-cell non-Hodgkin's lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic B-cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, multiple myeloma, marginal zone B-cell lymphoma, post-transplant lymphoproliferative disorder (PTLD), HIV-associated lymphoma, Waldenstrom's macroglobulinemia or Waldenstrom's macroglobulinemia. In particular, the CD 20-positive cancer is B-cell non-Hodgkin's lymphoma.

[0169] The present invention relates not only to the therapeutic treatment of those patients suffering from any of the above mentioned cancers, but also to the treatment of the recurrence of these cancers as well as to prophylactic treatment, for example, the treatment of patients susceptible to or affected by said diseases.

[0170] As used herein, "non-malignant disease" refers to any disease that does not fall within the above-mentioned cancer definition given. Non-malignant diseases that can be treated with the liquid pharmaceutical preparation of the present invention or HCLF include diseases selected from the following: autoimmune diseases, blood coagulation disorders, platelet diseases, hemoprotein disorders, blood diseases, hematological disorders, hemorrhagic disorders, coagulation disorders, lymphatic diseases, purpura, thrombocytopenia, thrombotic microangiopathy, coagulation disorders, vascular diseases, rheumatic diseases, connective tissue diseases, herpes virus infections and DNA virus infections. In particular, the liquid pharmaceutical preparation of the present invention or HCLF can be used to treat autoimmune diseases.

[0171] As used herein, "autoimmune disease" or "autoimmune-related disorder" refers to any disease in which the body makes an inappropriate immune response to tissues and / or substances naturally present in the body. Autoimmune diseases or autoimmune-related disorders include arthritis (e.g., rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), psoriasis, dermatitis (e.g., atopic dermatitis), chronic autoimmune urticaria, polymyositis / dermatomyositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, inflammatory bowel disease (e.g., Crohn's disease, ulcerative colitis), infant respiratory distress syndrome, adult respiratory distress syndrome (ARDS), meningitis, allergic rhinitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic disorders, eczema, asthma, disorders thereof Involved in T cell infiltration, and chronic inflammatory reactions, atherosclerosis, autoimmune myocarditis, leukocyte adhesion defects, systemic lupus erythematosus (SLE), lupus (e.g., lupus nephritis, non-renal lupus, discoid lupus, lupus alopecia), juvenile-onset diabetes, multiple sclerosis, allergic encephalomyelitis, immune responses associated with cytokine and T lymphocyte-mediated acute and delayed hypersensitivity reactions, tuberculosis, sarcoidosis, granulomas (including Wegener's granulomatosis), agranulocytosis, vasculitis (including ANCA), aplastic anemia, Coombs-positive anemia, Diamond Blackfan anemia, immune hemolytic anemia (including autoimmune hemolytic anemia (AIHA)), pernicious anemia, pure red cell aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, disorders involving leukocytopenia, CNS inflammatory disorders, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex-mediated disorders, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Behçet's disease, Castleman syndrome, Goodpasture syndrome, Lambert-Eaton myasthenic syndrome, Reynaud syndrome, Sjorgen syndrome, Stev ens-Johnson syndrome, solid organ transplant rejection (including pretreatment high group reactive antibody titer, IgA deposition in tissues, etc.), graft-versus-host disease (GVHD), bullous pemphigoid, pemphigus (including pemphigus vulgaris and pemphigus foliaceus), autoimmune polyendocrine disease, Reiter's disease, stiff-man syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathy or IgM-mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), autoimmune thrombocytopenia, autoimmune diseases of the testicle and ovary (including autoimmune orchitis and oophoritis), primary hypothyroidism;Autoimmune endocrinopathies, including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Graves' disease, autoimmune polyglandular syndrome (or polyglandular endocrinopathy syndrome), type I diabetes mellitus (also known as insulin-dependent diabetes mellitus (IDDM)), and Sheehan syndrome; autoimmune hepatitis, lymphocytic interstitial pneumonitis (HIV), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, large-vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu) arteritis), medium-vessel vasculitis (including Kawasaki disease and polyarteritis nodosa), ankylosing spondylitis, Berger's disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, sprue (gluten enteropathy), cryoglobulinemia, ALS, and coronary artery disease. In particular, the liquid pharmaceutical formulation or the HCLF is used to treat a disease selected from arthritis, thrombocytopenic purpura and / or systemic lupus erythematosus. In one embodiment, the liquid pharmaceutical formulation is used to treat arthritis, optionally rheumatoid arthritis. In another embodiment, the liquid pharmaceutical formulation is used to treat thrombocytopenic purpura. In another embodiment, the liquid pharmaceutical formulation is used to treat systemic lupus erythematosus. ;

[0172] As used herein, "inflammatory disease" refers to any disease involving acute or chronic inflammation, including inflammatory disorders such as allergies, asthma, cancer, and autoimmune diseases.

[0173] In one embodiment, the liquid pharmaceutical formulation or HCLF of the present invention can be used to treat a disease selected from leukemia, lymphoma and / or autoimmune disease.

[0174] In another embodiment, the liquid pharmaceutical formulation or HCLF of the present invention can be used to treat a disease selected from the group consisting of Burkitt's lymphoma, Epstein-Barr virus infection, B-cell leukemia, chronic lymphocytic B-cell leukemia, acute lymphocytic leukemia, lymphatic leukemia, prolymphocytic leukemia, hairy cell leukemia, multiple myeloma, B-cell lymphoma, marginal zone B-cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, immunoblastic large cell lymphoma, mantle cell lymphoma, non-Hodgkin's lymphoma, lymphomatoid granuloma, plasmacytoma, precursor cell lymphocytic leukemia-lymphoma, tumor virus infection, Waldenstrom's macroglobulinemia disease, rheumatoid arthritis, immunoproliferative disorders, prolymphocytic lymphoma, diffuse large B-cell lymphoma, immunoblastic large cell lymphoma, mantle cell lymphoma, lymphomatoid granuloma, lymphoproliferative disorders, atypical proteinemias, precursor cell lymphocytic leukemia-lymphoma, thrombocytopenic purpura, idiopathic thrombocytopenic purpura, blood coagulation disorders, platelet diseases, hemoproteinopathies, blood diseases, bleeding disorders, coagulation disorders, lymphatic diseases, purpura, thrombocytopenia, thrombotic microangiopathy, coagulation disorders, vascular diseases, rheumatoid arthritis, rheumatic diseases, connective tissue diseases, herpes virus infections and / or DNA virus infections.

[0175] The liquid pharmaceutical formulation or HCLF of the present invention can be administered to the patient subcutaneously or by other parenteral routes. Other parenteral routes include administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and / or surgical implantation at a specific site.

[0176] However, subcutaneous administration is particularly desirable. One advantage provided by subcutaneous injection is that it can be performed in a short time, especially when compared to intravenous injection (e.g., subcutaneous administration for about 10 minutes versus about 1 hour for intravenous infusion). Another advantage is that intravenous administration requires intravenous access, which must be established by trained personnel, while subcutaneous injection can even be performed by the patient himself, for example by using an automatic injection device, making the therapy more convenient for the patient.

[0177] Subcutaneous (SC) administration can be performed via a syringe (optionally pre-filled syringe), a pen injector (optionally automatic pen injector), an injection device or an infusion pump or a suitable needle-free device. Subcutaneous administration can be performed at a single site on the body or at different sites on the body, such as sites adjacent to each other. Suitable sites for subcutaneous administration are known to those skilled in the art and include, for example, the thigh or upper arm.

[0178] Subcutaneous injections are usually limited to a volume of about 2 ml or less. This is due to viscoelastic tissue resistance and back pressure (generated during injection) and the pain felt by the patient. Therefore, since a single subcutaneous injection can only provide a small volume of antibody preparation, it may be advantageous to have a preparation containing at least 160 mg / mL of an anti-CD 20 antibody, such as veltuzumab antibody. This allows the administration of high doses of antibodies with a small liquid volume suitable for subcutaneous injection.

[0179] The liquid pharmaceutical formulation or HCLF of the present invention may be provided in unit dose form (eg in an ampoule or a prefilled syringe or pen injector) or in multi-dose form (eg in a multi-dose container or an infusion pump).

[0180] The liquid pharmaceutical preparation or HCLF of the present invention may be administered alone or in combination with any other therapeutic agent deemed suitable for treating any of the above-mentioned diseases by a person skilled in the art. Said other therapeutic agent may be administered alone, simultaneously or sequentially with the preparation according to the present invention. Examples of such other therapeutic agents are, for example, cytotoxic agents, anti-angiogenic agents, corticosteroids, antibodies, chemotherapy, hormones, anti-inflammatory drugs and immunomodulators.

[0181] Examples of such therapeutic agents that can be administered alone or in combination with the liquid pharmaceutical formulation can be selected from antimitotic agents, antikinase agents, alkylating agents, antimetabolites, antibiotics, alkaloids, antiangiogenic agents, apoptotic agents, and combinations thereof. Chemotherapeutic agents can be selected, for example, from drugs such as vinca alkaloids, anthracyclines, epipodophyllotoxins, taxanes, antimetabolites, alkylating agents, antikinase agents, antibiotics, Cox-2 inhibitors, antimitotic agents, antiangiogenic agents, and apoptotic agents, particularly doxorubicin, methotrexate, paclitaxel, CPT-II, camptothecin, and other drugs from these and other classes of anticancer agents.

[0182] Further examples of such therapeutic agents are described, for example, in International Patent Application WO 2003 / 068821, the contents of which are incorporated herein and expressly referenced.

[0183] The present invention is further described in the following examples, which are merely for the purpose of illustrating specific embodiments of the invention and are not to be construed as limiting the scope of the invention in any way. Example

[0184] Example 1 Preparation of highly concentrated liquid formulations The drug substance bulk of veltuzumab was concentrated and diafiltered into the final buffer system via tangential flow filtration (TFF). An exemplary drug substance bulk comprises 60 g / L veltuzumab, 10 mM histidine, 120 mM sucrose, pH 5.5, such as Figure 2 The final drug substance bulk was then sterile filtered and stored below -40°C.

[0185] The excipients used in the present invention are generally of high purity and quality conforming to pharmacopoeia specifications (eg, European Pharmacopoeia).

[0186] The preparation of the highly concentrated liquid formulation is carried out by tangential flow filtration using a 30 kDa membrane. First, the final bulk buffer is exchanged with the new formulation buffer system according to the present invention. This diafiltration (DF) step is exemplarily shown in Figure 2 Typically, 8 volume exchanges are performed to remove the original buffer. In the next step (UF, ultrafiltration), the solution is concentrated to approximately 285 g / L of veltuzumab. The filter unit is then rinsed with diafiltration buffer to minimize losses. The concentration after rinsing is approximately 200 g / L. The remaining excipients are then added as a stock solution. In the case of mAb drug product mixtures, the final concentration is adjusted to 160 g / L by diluting with formulation buffer, such as Figure 2 Depicted.

[0187] All formulations at the drug product mixture stage were then sterile filtered through a filter with a pore size of 0.2 μm and filled into 2 mL glass vials under aseptic conditions. The vials were stoppered with ETFE-coated rubber stoppers and sealed with aluminum screw caps.

[0188] The different formulations were subjected to various stress conditions: such as elevated temperature (25 and 40°C), mechanical stress (shaking for 24h) and freeze-thaw stress. In addition, all samples were tested for long-term stability at refrigerated temperatures (2-8°C).

[0189] The samples were analyzed by the following analytical methods before and after applying the stress test.

[0190] Table 1 Overview of analytical methods

[0191] A further exemplary method for providing a high concentration liquid formulation of an antibody according to the present invention is Figure 1 and 3 Depicted in.

[0192] Example 2 Comparison of two highly concentrated liquid formulations of veltuzumab: Formulation A (phosphate-citrate-mannitol buffer) and Formulation B (histidine-sorbitol buffer) The following preparations were compared after storage at 25°C for three months: Formulation A: 150 mg / mL veltuzumab, 12 mg / mL mannitol, 6.2 mg / mL sodium chloride, 2.3 mg / mL sodium dihydrogen phosphate heptahydrate, 0.76 mg / mL sodium dihydrogen phosphate monohydrate, 1.3 mg / mL citric acid monohydrate, 0.34 mg / mL sodium citrate dihydrate, 1.0 mg / mL polysorbate 80, pH 5.2 Formulation B: 150 mg / mL veltuzumab, 50 mg / mL sorbitol, 30 mM L-histidine, 200.1 mg / mL polysorbate, appropriate amount of acetic acid, pH 5.2.

[0193] Table 2 Comparison of formulations A and B after storage at 25°C for three months

[0194] As can be obtained from Table 2, Formulation B, which is a formulation according to the invention comprising a histidine buffer system, is clearly superior to Formulation A, especially considering the parameters: aggregation, fragmentation and effective concentration. Assay, microscopic particles and potency are within comparable ranges.

[0195] It was thus clearly shown that histidine not only acts as a buffer substance, but also stabilizes veltuzumab at higher concentrations.

[0196] Example 3 Formulation Optimization Regarding Histidine and pH Formulation B defined in Example 2 was optimized using long term storage conditions.

[0197] In particular, the following two factors were investigated: histidine concentration (10 to 50 mM) and pH (4.8 to 6.2).

[0198] The responses are the analytical parameters as shown in Table 5. A full factorial statistical design of experiment using the software Modde (Umetrics) was used. Aggregation was shown to be the only analytical parameter (response) significantly affected by two factors, histidine concentration and pH. All other responses were not affected by factors within the experimental range.

[0199] Figure 4 The response to aggregation is depicted in the contour plot. At relatively low pH and high histidine concentrations, the aggregation tendency can be slowed down. However, it can be shown in another set of experiments (not shown here) that histidine concentrations above 50 mM (estimated to be up to 100 mM) no longer reduce aggregation further. It is further considered that for subcutaneous formulations, the pH should be close to the physiological pH of 7.4. The concentration of sorbitol can therefore be adjusted to provide the physiological tonicity of the solution.

[0200] Example 4 Preparation optimization of polysorbate 20 concentration It is generally known that protein solutions tend to form insoluble aggregates upon mechanical stress. This phenomenon is usually explained as surface-induced protein degradation and can be reduced by introducing non-ionic surfactant ingredients such as polysorbates into the formulation.

[0201] Formulation C (veltuzumab 160 g / L, sorbitol 40 g / L, L-histidine 30 mM, pH 5.5) was used in these examples to determine what concentration of polysorbate 20 would be needed to stabilize the liquid formulation against microparticle formation during mechanical and freeze-thaw stress. Four levels of polysorbate 20 were tested: 0; 0.1, 0.2, and 0.3 g / L.

[0202] Table 3 Formulation C and different polysorbate 20 concentrations Veltuzumab 160mg / mL Sorbitol 40mg / mL. L-Histidine 30mM pH 5.5 Polysorbate 20 0; 0.1; 0.2; 0.3 mg / mL .

[0203] The results are summarized in the following table: Table 4 Comparison of Formulation C with different levels of polysorbate 20

[0204] As expected, the addition of polysorbate 20 had a large effect on the formation of (microscopic) microparticles as measured by microfluidic imaging (MFI). The microparticle counts increased only slightly after freeze-thaw and 0.1 mg / mL of polysorbate 20 was sufficient to prevent microparticle formation. However, the microparticle counts were much higher after shaking stress. The addition of 0.1 mg / mL of polysorbate 20 was not sufficient to completely avoid microscopic microparticles in the solution.

[0205] Four formulations with different levels of polysorbate 20 were also placed under long-term storage conditions (2-8°C) to verify the compatibility of veltuzumab with surfactants during normal storage. After 12 months of storage, it was apparent that the polysorbate levels studied had no effect on any of the quality attributes (parameters) tested. Therefore, 0.2 mg / mL of polysorbate 20 appears to be the ideal amount to stabilize veltuzumab against microparticle formation (optimized formulation C).

[0206] Example 5 Stability testing of different antibody concentrations in the formulation Different concentrations of veltuzumab were tested in optimized formulation C (veltuzumab 160 / 190 / 220 g / L, sorbitol 40 g / L, L-histidine 30 mM, pH 5.5, polysorbate 200.2 g / L), and the stability was determined over specific time periods under different temperature and humidity conditions.

[0207] The stability results in Tables 5 to 11 show acceptable values ​​for all tested concentrations of veltuzumab in optimized formulation C for the observed time periods, temperatures, and relative humidity, respectively.

[0208] Example 6 Patients were treated with veltuzumab in optimized formulation C A randomized, double-blind, placebo-controlled, multicenter, multinational, phase II, 4-arm, parallel-group trial was conducted to compare three different subcutaneous (sc) doses of the anti-CD20 monoclonal antibody veltuzumab with placebo as add-on therapy to methotrexate in subjects with moderate to severe rheumatoid arthritis (RA) inadequately controlled with methotrexate (MTX) or methotrexate plus anti-tumor necrosis factor (anti-TNF) biologic therapy. Four hundred subjects were screened to allow 320 eligible subjects to be randomly assigned in a 1:1:1:1 ratio to one of the four treatment arms (80 subjects per arm).

[0209] Veltuzumab is a humanized monoclonal IgG1 antibody targeting the CD20 epitope, which is ubiquitously expressed on the surface of mature B cells, leading to their depletion. Veltuzumab is administered by subcutaneous injection without steroid premedication and is expected to offer improved convenience and cost-effectiveness compared to other anti-CD20 therapies.

[0210] The trial was designed to compare veltuzumab at three different dose levels (160 mg, 320 mg, and 640 mg) with placebo, administered once a week for four weeks (days 1, 8, 15, and 22) by subcutaneous injection to moderate to severe RA subjects (the cumulative veltuzumab doses were 640 mg, 1280 mg, and 2560 mg, respectively). All subjects continued to be co-administered with methotrexate in a stable manner. The primary endpoint, the American College of Rheumatology 20 (ACR20) response rate, was assessed at week 24.

[0211] Subcutaneous veltuzumab is expected to be generally well tolerated. No clinically significant observations or treatment-related serious adverse events are expected.

[0212] The total volume administered subcutaneously to an individual patient is 2.0 mL per injection. In summary, subcutaneous veltuzumab is expected to be delivered quickly, comfortably and safely to patients. The patient experience is expected to be good, which is a supportive test for veltuzumab in Phase III trials.

Claims

1. A method for preparing an antibody having a concentration of C H A method for preparing a high concentration liquid formulation of an antibody, the method comprising the steps of: a) provide a starting concentration of C S A solution of antibodies and histidine; b) ultrafiltration of the solution of step (a) to obtain a solution having an intermediate antibody concentration C I A solution of which C I Up to 285 mg / mL; and c) diluting the solution of step (b) with a buffer solution containing histidine, polysorbate and sorbitol to an antibody concentration of C H To obtain a high concentration liquid formulation with a pH value of 5.5, wherein C H is 160 mg / mL, 190 mg / mL or 220 mg / mL, and the antibody is veltuzumab, the concentration of histidine is in the range of 30 mM, the polysorbate is polysorbate 20 in the range of 0.2 mg / mL, and the concentration of sorbitol is 220 mM.

2. The method according to claim 1, wherein the concentration C of veltuzumab H It is 160mg / ml.

3. The method according to claim 1, wherein the concentration C of veltuzumab H It is 190mg / ml.

4. The method according to claim 1, wherein the concentration C of veltuzumab is H It is 220mg / ml.

5. The method according to any one of claims 1 to 4, further comprising a step of diafiltration of the solution of step (a) with a buffer solution between step (a) and step (b), wherein the buffer is histidine.

6. The method according to any one of claims 1 to 4, wherein the solution subjected to ultrafiltration in step (b) contains 40 mM histidine and has a pH value of 5.

45.

7. The process according to any one of claims 1 to 4, wherein the solution subjected to ultrafiltration in step (b) does not contain a tonicity modifier.

8. The process according to any one of claims 1 to 4, wherein the solution subjected to ultrafiltration in step (b) does not contain sucrose.

9. The process according to any one of claims 1 to 4, wherein the solution subjected to ultrafiltration in step (b) does not contain a surfactant.

10. The process according to any one of claims 1 to 4, wherein the solution subjected to ultrafiltration in step (b) does not contain polysorbate.

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