WO2014045081A1

WO2014045081A1 - Stable pharmaceutical composition, comprising an aqueous solution of an antibody-derived therapeutically active protein

Info

Publication number: WO2014045081A1
Application number: PCT/IB2012/054950
Authority: WO
Inventors: Olivier Soula; Thomas Ballet
Original assignee: Adocia
Priority date: 2012-09-18
Filing date: 2012-09-18
Publication date: 2014-03-27

Abstract

The invention concerns a storage-stable pharmaceutical composition, comprising an aqueous solution of: (a) At least an antibody-derived therapeutically active protein chosen amongst antibody, nanobody or fusion protein; (b) an amount effective to stabilize said antibody-derived therapeutically active protein of at least one lauryldimethylamineoxide and/or of one of its amine oxide analogs.

Description

Stable pharmaceutical composition, comprising an aqueous solution of an antibody-derived therapeutically active protein.

[0001] The present invention relates generally to compositions and methods thereof that increase physical stability by reducing or preventing aggregation of antibody- derived proteins in therapeutically useful formulations, and specifically, to compositions having at least an antibody-derived therapeutic protein and at least lauryldimethylamineoxide and/or one of its alkylamine oxide analogs.

[0002] Like most of protein products, antibody-derived therapeutics proteins are known to be unstable in liquid formulations. Indeed, proteins undergo numerous physica! and chemical changes that affect potency and safety. Among these are aggregation, which includes formation of dimers, trimers, and higher-order protein aggregates as described in Mahler, H.C. et al. Induction and Analysis of Aggregates in a Liquid IgGl-Antibody Formulation. Eur.J.Pharm.Biopharm. 2005, 59 (3), 407-417 or in Mahler, H.C. et al. Protein Aggregation : Pathways, Induction Factors and Analysis. J Pharm.Sci. 2009, 98 (9), 2909-2934.

[0003] Aggregation may occur during the course of manufacturing process or during storage because of high-shear and mechanical stresses. Furthermore, the delivery of high protein concentration is often required for parenteral administration, especially for subcutaneous injection, due to dose requirements (usually > 50 nrtg/mL, preferably > 100 mg/mL) and to the volume limitations ( 1.5 mL). Such concentrated protein solutions generate several problems, including the tendancy of proteins to aggregate during processing and/or storage, because of the increased likelihood of protein- protein interactions in concentrated regimen.

[0004] In an attempt to obviate the physical stability problem, protein formulations have been formulated and commercialized in a dry form, i.e. in a lyophilized form. For example, stable lyophilized protein formulations are disclosed in PCT publication WO 97/04801. The disclosed lyophilized formulations can be reconstituted to generate high protein-concentration liquid formulations without apparent loss of stability. However, the convenience of administration and improved patient compliance make a stable liquid formulation a better choice than a lyophilized product.

[0005] In the case of liquid formulations, the physical stability problem is usually circumvented by adding a suitable surfactant, most of the time polysorbate 20 or polysorbate 80. Polysorbates are the most widely used non-ionic surfactants to stabilize protein pharmaceuticals against interface-induced aggregation and surface adsorption.

[0006] They have been shown to be quite effective against various stresses such as agitation, freeze/thawing and lyophilization. Nevertheless, some of their characteristics need to be carefully considered and monitored. Indeed, commercially available polysorbates 20 and 80 are chemically diverse mixtures containing mainly sorbitan polyoxyethylene (POE) fatty acid esters.

[0007] Additionally, substantial amounts of POE, sorbitan POE and isosorbide POE fatty acid esters are present. This leads to a significant degree of lot-to-!ot variability requiring a close scrutiny of each lot in order to ensure uniform behavior. The presence of residual levels of peroxide in bulk polysorbate is also a concern.

[0008] The European Pharmacopeia specifies a limit for peroxide number < 10, There have been reports of a buildup of peroxides in bulk as well as in aqueous solutions of polysorbate, when exposed to ambient oxygen and light.

[0009] Depending on handling and storage conditions and supplier lot, varying concentrations of peroxides (9 ppm- 250 ppm) were noted among different lots of polysorbates (See for example Kishore, R.S. et al. Degradation of Polysorbates 20 and 80: Studies on Thermal Autoxidation and Hydrolysis. J Pharm.Sci. 2010. or Kishore, R.S. et a!. The Degradation of Polysorbates 20 and 80 and Its Potential Impact on the Stability of Biotherapeutics. Pharm.Res 2011, 28 (5), 1194-1210).

[00010] The buildup of peroxides can be detrimental not only to the stability of polysorbate itself but also to the therapeutic protein, which it stabilizes. For example, it has been described in the literature that certain polysorbate concentrations result in increased protein aggregation (See Kiese, Ξ. et ai. Shaken, Not Stirred : Mechanical Stress Testing of an IgGl Antibody. J Pharm.Sci. 2008, 97 (10), 4347-4366).

[00011] The present invention relates to a method to formulate storage-stable pharmaceutical compositions, comprising an aqueous solution of a therapeutically effective proteins/peptides such as an antibody, a nanobody or a fusion protein-based on the surprising discovery that lauryldimethyiamineoxide and/or of one of its amine oxide analogs had the ability to stabilize aqueous solutions of therapeutically effective proteins/peptides.

[00012] The present invention relates to the use of one lauryldimethyiamineoxide and/or of one of its amine oxide analogs to stabilize an antibody-derived therapeutically active protein in aqueous solutions and to the storage-stable pharmaceutical compositions obtained.

[00013] Surprisingly, the aqueous solutions are better stabilized than by adding polysorbates and the stabilizing effect is independent of the pH and tonic strength.

[00014] Moreover the active concentrations of the lauryldimethyiamineoxide and/or of one of its amine oxide analogs are independent of the antibody-derived therapeutically active protein concentrations and can be lower than that of other surfactants which make them attractive for highly concentrated protein stabi!ization.

[00015] In one embodiment the invention relates to a method of providing storage stability to an aqueous pharmaceutical formulation of an antibody-derived therapeutically active protein such as antibody, nanobody or fusion protein comprising admixing under steriie conditions in an aqueous solution an antibody-derived therapeutically active protein and an amount effective to stabilize said antibody- derived therapeutically active protein of at least one lauryldimethylamineoxide and/or of one of its amine oxide analogs.

[00016] In another embodiment the invention relates to a method of increasing the shelf life of an aqueous pharmaceutical formulation of an antibody-derived therapeutically active protein such as antibody, nanobody or fusion protein comprising admixing under sterile conditions in an aqueous solution an antibody-derived therapeutically active protein and an amount effective to stabilize said antibody- derived therapeutically active protein of at least one lauryldimethylamineoxide and/or of one of its amine oxide analogs.

[00017] The invention also relates to the use of at least one lauryldimethylamineoxide and/or of one of its amine oxide analogs to stabilize a stored, aqueous pharmaceutical formulation of an antibody-derived therapeutically active protein such as an antibody, a nanobody or a fusion protein,

[00018] The invention also relates to a storage-stable pharmaceutica! composition, comprising an aqueous solution of:

a) At least an antibody-derived therapeutically active protein chosen amongst antibody, nanobody or fusion protein;

b) an amount effective to stabilize said antibody-derived therapeutically active protein of at least one laury!dimethy!amineoxide and/or of one of its amine oxide analogs.

[00019] The stable liquid formulations of antibody-derived therapeutically active protein exhibit stability, low to undetectable levels of antibody fragmentation and/or aggregation, and very little to no ioss of the biological activities of the antibodies (including antibody fragments thereof) during manufacture, preparation, transportation, and storage. Stability can be assessed by, for example, visual inspection, or high performance size exclusion chromatography (HPSEC). The stability of the antibody formulations may be measured by, for example, high performance size exclusion chromatography (HPSEC). The techniques of static tight scattering (SLS), Fourier Transform Infrared Spectroscopy (FTIR), circular dichroism (CD), intrinsic tryptophan fluorescence, differentia! scanning caiorimetry, and/or ANS protein binding are also used to assess the phase behaviors, other physical properties and stability of the antibody molecules. Visual or spectroscopic inspection of aggregation is a preferred manner of determining stability.

[00020] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains additional components, i.e excipients which are pharmaceutically acceptable.

[00021] A "stable" or "stabilized" formulation is one in which the antibody-derived therapeutically active protein such as antibody, nanobody or fusion protein retains its physical and/or chemical stability upon storage. Stability can be measured at a selected temperature for a selected time period. Preferably, the formulation is stable at room temperature or at 40°C for at least 1 month and/or stable at about 2-8°C for at least 1 year and preferably for at least 2 years. The aggregation during storage is used as an indicator of protein stability.

[00022] Thus, a "stable" formulation may be one wherein less than about 10% and preferably less than about 5% of the protein is present as an aggregate in the formulation,

[00023] The "stable" formulations of the invention retain biological activity under given manufacture, preparation, transportation and storage conditions. The antibody formulations of the invention maintain improved aggregation profiles upon storage, for example, for extended periods (for example, but not limited to 6 months, 1 year, 2 years, 3 years or 5 years) at room temperature or 2-8°C or for periods (such as, but not limited to 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months or 1 year) at elevated temperatures such as 38°C-42°C.

[00024] In one aspect of the invention, the method provides an antibody-derived therapeutically active protein formulation with low to undetectable levels of aggregation. The phrase "low to undetectable levels of aggregation" as used herein refers to samples containing no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1% and no more than 0.5% aggregation by weight of protein as measured by high performance size exclusion chromatography (HPSEC), static light scattering (SLS), Fourier Transform Infrared Spectroscopy (FTIR), circular dichroism (CD), intrinsic tryptophan fluorescence, differential scanning caiorimetry, and l-anilino-8-naphthalenesulfonic acid (ANS) protein binding techniques. Furthermore, liquid formulations of the present invention exhibit almost no loss in biological activities of the antibody (including antibody fragment thereof) during the prolonged storage under the condition described above, as assessed by various immunological assays including, for example, enzyme-iinked immunosorbent assay (ELISA) and radioimmunoassay to measure the ability of the antibody (including antibody fragment thereof) to immunospecifically bind to an antigen. The liquid formulations of the present invention retain after the storage for the above-defined periods more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99%, or more than 99.5% of the initial biological activities.

[00025] The term "aqueous solution" refers to a solution in which water is the dissolving medium or solvent. When a substance dissolves in a liquid, the mixture is termed a solution. The dissolved substance i.e. the antibody-derived therapeutically active protein such as antibody, nanobody or fusion protein is the solute, and the liquid that does the dissolving (in this case water) is the solvent.

[00026] In a specific embodiment the antibody-derived therapeutically active protein is an antibody.

[00027] As used herein, the term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a therapeutic target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing, The term "antibody" is referred to herein to encompass full-length monoclonal or polyclonal antibodies, as well as antibody fragments, such as Fab, Fab', F(ab')2, and Fv fragments, single chain Fv (scFv) mutants and Fc fusion proteins. Therapeutic antibodies of the invention include multispecific antibodies such as bispecific antibodies generated from at least two full-length antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins, peptibodies comprising an antigen recognition portion or an Fc portion of an antibody, and modified immunoglobulin molecules comprising an antigen recognition site (or portion thereof) or Fc domain, so long as the protein exhibits the desired biological (e.g., therapeutic) activity. A therapeutic antibody can be of any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations. Therapeutic antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[00028] The term "antibody fragment" refers to a portion of an antibody that includes an antigen recognition site (or portion thereof) and/or non-antigen recognition site, such as an effector domain, as well as variants or derivatives of an antibody. Thus, "antibodies" include polypeptides containing an antibody fragment that has an effector domain and/or all or a portion of an antigen recognition site. Antibody fragments include single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and Fc fusion proteins. Antibody fragments can be of any type (e.g., IgG, IgE, Ig , IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule. Antibody fragments, including single-chain antibodies, may comprise one or more CDR regions alone (e.g., CDR2 and CDR3) or any combination of CDR regions, the variable region(s) alone or in combination with the entirety or a portion of the following : hinge region, CHI, CH2, and CH3 domains. Antibody fragments can contain any combination of variable region(s) with a hinge region, CHI, CH2, and CH3 domains. Antibody fragments may additionally, or alternatively, include portions of an antibody constant region that confer antibody effector function (e.g., immunoglobulin effector domain sequences) or that mediate antibody half-iife. In particular embodiments, therapeutic antibodies of the invention comprise an Fc domain. In additional embodiments, the antibodies are Fc fusion proteins. Antibody fragments can be from any appropriate source and may be of synthetic (e.g., chimeric, humanized and otherwise modified antibodies) or animal origin (e.g., birds and mammals). Antibody fragments that recognize specific epitopes and/or that compete for target binding with another antibody or protein can be generated, identified, and characterized using techniques known in the art.

[00029] The terms "therapeutic antibody" and "therapeutically effective antibody" are used interchangeably herein and refer to an antibody that when administered to a subject in a sufficiently effective amount, prevents, delays, alleviates or arrests the onset and/or further development of the symptoms, complications, or biochemical indicia of a disease, condition, or disorder in the subject (e.g., a patient such as a human patient, non-human primate, or an experimental animal such as a rabbit, rat, mouse or other animal).

[00030] The terms "compete," "compete for binding" and "competes with" are relative terms used to describe an antibody that produces a 50% inhibition of binding to a target by an antibody or reference cognate ligand as determined in a standard competition assay as described herein or otherwise known in the art, including, but not limited to, competitive assay systems using techniques such as radioimmunoassays (RIA), enzyme immunoassays (EIA), preferably the enzyme iinked immunosorbent assay (ELISA), "sandwich" immunoassays, immunoradiormetric assays, fluorescent immunoassays, luminescent, electrochemical luminescent, and Immunoelectrophoresis assays. Methods for determining binding and affinity of candidate binding molecules are known in the art and include, but are not limited to, affinity chromatography, size exclusion chromatography, equilibrium dialysis, fluorescent probe displacement, and plasma resonance.

[00031] An antibody is said to "competitively inhibit" binding of a reference molecule to a given epitope if it binds to that epitope to the extent that it blocks, to some degree, binding of the reference molecule to the epitope, Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays. As used herein, an antibody can be said to competitively inhibit binding of the reference molecule to a given epitope, for example, by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[00032] In some embodiments, the therapeutically active antibody is a member selected from : Muromonab-CD3 (product marketed with the brand name Orthoclone Okt3®), Abciximab (product marketed with the brand name Reopro®), Rituximab (product marketed with the brand name MabThera®, Rituxan®), Basifiximab (product marketed with the brand name Simulect®), Daclizumab (product marketed with the brand name Zenapax®), Palivizumab (product marketed with the brand name Synagis®), i'lnfliximab (product marketed with the brand name Remicade®), Trastuzumab (product marketed with the brand name Herceptin®), Gemtuzumab ozogamicin (product marketed with the brand name Mylotarg®), Alemtuzumab (product marketed with the brand name MabCampath®, Campath-1H®), Adalimumab (product marketed with the brand name Humira®), Tositumomab-1131 (product marketed with the brand name Bexxar®), Efalizumab (product marketed with the brand name Raptiva®), Cetuximab (product marketed with the brand name Erbitux®), ibritumomab tiuxetan (product marketed with the brand name Zevalin®), I'Omalizumab (product marketed with the brand name Xoiair®), Bevacizumab (product marketed with the brand name Avastin®), Natalizumab (product marketed with the brand name Tysabri®), Ranibizumab (product marketed with the brand name Lucentis®), Panitumumab (product marketed with the brand name Vectibix®), I'Eculizumab (product marketed with the brand name Soliris®), Certolizumab pegol (product marketed with the brand name Cimzia®), Golimumab (product marketed with the brand name Simponi®), Canakinumab (product marketed with the brand name Ilaris®}, Catumaxomab (product marketed with the brand name Removab®), I'Ustekinumab (product marketed with the brand name Ste!ara®), Tocilizumab (product marketed with the brand name RoActemra®, Actemra®), I'Ofatumumab (product marketed with the brand name Arzerra®}, Denosumab (product marketed with the brand name Prolia®), Belimumab (product marketed with the brand name Beniysta®), Raxibacumab, Ipi!imumab (product marketed with the brand name Yervoy®), Brentuximab vedotin (product marketed with the brand name Adcetris®) and Pertuzumab (product marketed with the brand name Perjeta®).

[00033] In a specific embodiment, the antibody-derived therapeutically active protein is a fusion protein.

[00034] As used herein, the term "fusion proteins" by itself or as part of another phrase, refers to proteins created through the joining of two or more genes which originally coded for separate proteins/peptides. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins. Recombinant fusion proteins are created artificially by recombinant DNA technology for use in biological research or therapeutics. Chimeric mutant proteins occur naturally when a complex mutation, such as a chromosomal translocation, tandem duplication, or retrotransposition creates a novel coding sequence containing parts of the coding sequences from two different genes. Naturally occurring fusion proteins are commonly found in cancer cells, where they may function as oncoproteins.

In one embodiment, the therapeutically active fusion protein is a member selected from : Abatacept (product marketed with the brand name Orencia®), Etanercept (product marketed with the brand name Enbrel®), Rilonacept (product marketed with the brand name Arcalyst®) and Aiefacept (product marketed with the brand name Amevive®).

[00035] In a specific embodiment the antibody-derived therapeutically active protein is a nanobody.

[00036] As used herein, the term "nanobody" or "single domain antibody" refers to an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. Nanobodies are described in D. Saerens and Ξ. Muyldermans (eds.) Single Domain Antibodies : Methods and Protocols, Methods in Molecular Biology, vol. 911; and Med Microbiol Immunol (2009). [00037] By "amine oxide analogs of laury!dimethylamineoxide" it is meant amine oxide chosen amongst the amine oxides of formula I:

Formula I wherein

m represents an integer comprised in the interval from 0 to 17, 0 ≤ m < 17,

n represents an integer comprised in the interval from 0 to 17, 0 < n < 17, a represents an integer equal to 0 or 1,

9 < n + m < 17

If m=0, then a=0

F represents a function chosen in the group constituted by the functions amide, ester, carbamate and urea,

Rl and R2, identical or different, represent alkyl chains comprising from 1 to 4 carbon atoms.

[00038] In a further embodiment, the analogs of lauryldimethy!amineoxide are chosen amongst the amine oxide analogs of formula I, wherein n represents an integer comprised in the interval from 9 to 13, 9 =n≤ 13.

[00039] In a further embodiment, the analogs of iaury!dimethylamineoxide are chosen amongst the amine oxide analogs of formula I , wherein a = 0 and m = 0 and the amine oxide is chosen ds of formula II:

Formula II

wherein,

^■ n represents an integer comprised in the interval from 9 to 17, 9 < n < 17, « Rl and R2, identical or different, represent aikyi chains comprising from 1 to 4 carbon atoms. [00040] In a further embodiment, the analogs of lauryldimethyiamineoxide are chosen amongst the amine oxide analogs of formula II, wherein n represents an integer comprised in the interval from 9 to 13, 9 < n < 13.

[00041] In a further embodiment, the concentration of the at least one lauryldimethyiamineoxide and/or amine oxide analogs is from 0.01 mM to 100 mM.

[00042] In a further embodiment, the concentration of the at least one lauryldimethyiamineoxide and/or amine oxide analogs is from 0.1 mM to 10 mM.

[00043] In a further embodiment, the concentration of the at least one lauryldimethyiamineoxide and/or amine oxide analogs is from 0.5 mM to 1.5 mM,

[00044] In a further embodiment, the at least one amine oxide analog is N,M - Dimethyldecylamine N-oxide (CAS 2605-79-0).

[00045] In a further embodiment, the at least one amine oxide analog is Ν,ΙΜ - Laury!dimethylamine N-oxide (CAS 1643-20-5).

[00046] In a further embodiment, the at least one amine oxide analog is ΙΜ,Ν - Dimethyltetradecylamine N-oxide (CAS 3332-27-2).

[00047] In a further embodiment, the at least amine oxide analog is N-2- (dimethylnitrory!)ethyidodecanamide (CAS 86321-42-8).

[00048] In a further embodiment, the composition has a pH that is in the range from about 5.0 to 8.0.

[00049] In a further embodiment, the composition has a pH that is in the range from about 5.5 to 7.8.

[00050] In a further embodiment, the composition has an osmolality in the range from about 150 to 800 mOsm/kg.

[00051] In a further embodiment, the composition has an osmolality in the range from about 200 to 500 mOsm/kg,

[00052] In one embodiment at least one additional surfactant is added to the composition.

[00053] "Surfactants" are surface active agents that can exert their effect at surfaces of solid-solid, solid-liquid, liquid-liquid, and liquid-air because of their chemical composition, containing both hydrophilic and hydrophobic groups. [00054] In one embodiment of the invention, the at ieast one additional surfactant is chosen amongst polysorbate 20 (CAS 9005-64-5) and polysorbate 80 (CAS 9005-65- 6),

[00055] In another embodiment of the invention, the at Ieast one additional surfactant is chosen amongst n-Dodecyl β-D-maltoside { CAS 69227-93-6), Poiyoxyi 35 hydrogenated castor oil (Cremophor EL, CAS 61791-12-6), and Polyoxyethyiene- polyoxypropylene block copolymer (Pluronic F-68, CAS 9003-11-6).

[00056] In one embodiment the surfactant is a zwitterionic surfactant i.e. (Lauryldimethylammonio)acetate (S6, CAS 683-10-3, Sigma-Aldrich).

[00057] In one embodiment at least one additional viscosity reducer is added to the composition.

[00058] A "viscosity reducer" is a compound that is capable of measurably reducing viscosity of an aqueous protein-containing formulation. In one embodiment the viscosity reducer is arginine hydrochloride or one compound chosen amongst the compounds described in US provisional application in the name of Adocia n° 61/682003.

[00059] In one embodiment at Ieast one pharmaceutically acceptable acid is added to the composition.

[00060] A "pharmaceutically acceptable acid" includes inorganic and organic acids which are non toxic at the concentration and manner in which they are formulated. For example, suitable acids include hydrochloric, phosphoric, citric, acetic, ascorbic, ethylenediaminetetracetic acid (EDTA) and tartaric acids.

[00061] In one embodiment at ieast one pharmaceutically acceptable base is added to the composition.

[00062] "Pharmaceuticaily-acceptable bases" include inorganic and organic bases which are non-toxic at the concentration and manner in which they are formulated. For example, suitable bases include those formed from inorganic base forming metals such as sodium, potassium, magnesium, calcium and ammonium.

[00063] Additional pharmaceutically acceptable acids and bases useable with the present invention include those which are derived from the amino acids, for example, histidine, arginine, glycine, phenylalanine, lysine and asparagine.

[00064] In one embodiment at Ieast one buffer is added to the composition.

[00065] "Buffers" and salts include those derived from both acid and base addition salts of the above indicated acids and bases. [00066] In one embodiment at least one pharmaceutical diluent is added to the composition.

[00067] For example, suitable diluents include injection sterilized water, buffer water solutionand a IMaCI solution (NaCI 0.9%).

[00068] In one embodiment at least one pharmaceutical preservative is added to the composition.

[00069] For example, suitable preservative include benzy!ic alcohol, phenol, m-cresol and povidone.

[00070] In an additional embodiment, the composition of the pharmaceutical formulation is the same as or substantially similar to a formulation for administering the corresponding therapeutic antibody, fusion protein or nanobody that has been approved by a regulatory agency for administration to a subject, but that additionally contains lauryidimethylamineoxide and/or one or more of its amine oxide analogs. In particular embodiments, the lauryidimethylamineoxide and/or amine oxide analog(s) are at a concentration > CMC.

[00071] In an additional embodiment, the composition of the pharmaceutical formulation is the same as or substantially similar to a formulation for administering the corresponding therapeutic antibody, fusion protein or nanobody that has been approved by a regulatory agency for administration to a subject, but that additionally contains lauryidimethylamineoxide and/or one or more of its amine oxide analogs. In particular embodiments, the lauryidimethylamineoxide and/or amine oxide analog(s) are at a concentration > or equal to about 1.5 times its CMC.

[00072] The invention provides for pharmaceutical compositions for intravenous administration comprising at least about 1 mg/mL of an antibody-derived therapeutically active protein.

[00073] The invention provides for pharmaceutical compositions for intravenous administration comprising from 1 mg/mL to 50 mg/mL of an antibody-derived therapeutically active protein.

[00074] In additional embodiments, the invention provides for pharmaceutical compositions for subcutaneous administration comprising at least about 50 mg/mL of a therapeutic antibody, nanobody or fusion protein . [00075] In additional embodiments, the invention provides for pharmaceutical compositions for subcutaneous administration comprising from 50 mg/mL to 350 mg/mL of a therapeutic antibody, nanobody or fusion protein.

[00076] In additional embodiments, the invention provides for pharmaceutical compositions for subcutaneous administration comprising from 50 mg/mL to 250 mg/mL of a therapeutic antibody, nanobody or fusion protein.

[00077] In additional embodiments, the invention provides for pharmaceutical compositions for subcutaneous administration comprising from 50 mg/mL to 150 mg/mL of a therapeutic antibody, nanobody or fusion protein.

[00078] In additional embodiments, the invention provides for pharmaceutical compositions for subcutaneous administration comprising from 50 mg/mL to 100 mg/mL of a therapeutic antibody, nanobody or fusion protein.

[00079] In another embodiment, the invention provides for pharmaceutical compositions containing a concentration of antibody-derived therapeutically active protein that corresponds to a corresponding marketed or government approved therapeutic formulation for the therapeutic antibody-derived therapeutically active protein. In some embodiments the therapeutic antibody-derived therapeutically active protein concentration is about 5 mg/mL and the therapeutic antibody-derived therapeutically active protein is infliximab ( EMICADE®) or cetuximab (ERBITUX®). In some embodiments the therapeutic antibody-derived therapeutically active protein concentration is about 10 mg/mi and the therapeutic antibody-derived therapeutically active protein is selected from : bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®) and rituximab (RITUXAN®).

[00080] According to some embodiments the invention encompasses a method of administering a formulation of the invention to a patient comprising the steps of injecting an aqueous formulation that comprises at least an intravenous injection, subcutaneous injection or intramuscular injection.

[00081] The invention also concerns a pharmaceutical container, comprising a hermetically sealed vessel and the pharmaceutical composition as described above.

[00082] In one embodiment the invention, the pharmaceutical container, where in the vessel is a vial, bottle, pre-fi!led syringe or pre-filled auto-injector. Example 1 - Solution of Bevacizumab (Avastin®) at 10 mg/mL

[00083] The formulation at 25 mg/mL (pH 6.2) is marketed by Genentech/Hoffmann- La Roche. For the purpose of the experiment, this formulation was diluted to 10 mg/mL and was completed with its excipients in order to reach the following concentrations in the final solution : 158 mM of trehalose, 50 mM of sodium phosphate and 0.33 mM of polysorbate 20.

Example 2 - Solution of Infliximab (Re mi cade®) at 5 mg/mL

[00084] The formulation is a freeze-dried product reconstituted at 10 mg/mL with water (pH 7.2). In Europe, it is marketed by Schering-Plough. For the purpose of the experiment, this formulation was diluted to 5 mg/mL and was completed with its excipients in order to reach the following concentrations in the final solution : 146 mM of saccharose, 50 mM of sodium phosphate and 38 μΜ of polysorbate 80.

Example 3 - Solution of Trastuzumab (Herceptin®) at 10 mg/mL

[00085] The formulation is a freeze-dried product reconstituted at 21 mg/mL with water for injection (pH 6). In Europe, it is marketed by Hoffmann - La Roche. For the purpose of the experiment, this formulation was diluted to 10 mg/mL and was completed with its excipients in order to reach the following concentrations in the final solution : 2.3 mM of L-histidine, 52.8 mM of trehalose and 72 pm of polysorbate 20.

Example 4 - Solution of Rituximab (Mab Thera® or Rituxan®) at 10 mg/mL

[00086] The formulation at 10 mg/mL is marketed by Hoffmann - La Roche in Europe (pH 6.5). This formulation contains 153 mM of sodium chloride, 25 mM of sodium citrate and 534 μΜ of polysorbate 80. The commercial formulation was used as such.

Example 5 - Solution of Cetuximab (Erbitux®) at 75 mg/mL

[00087] The formulation at 5 mg/mL is marketed by Merck (pH 5.3-5.7) in Europe. This formulation contains sodium chloride, glycine, citric acid and polysorbate 80 in unknown amounts. For the purpose of the experiment, it was concentrated at 75 mg/mL. Example 6 - Solution of Abatacept (Orencia^®) at 25 mg/mL

[00088] The formulation is a freeze-dried product reconstituted at 25 mg/mL with water for injection (pH 7.2-7.8). It is marketed by Bristol-Meyer Squibb Pharma in Europe, Abatacept is a fusion protein composed of the Fc region of the immunoglobulin IgGl fused to the extracellular domain of CTLA-4. This solution contains 50 mg/mL of maltose, 1.7 mg/mL of sodium phosphate and 1.5 mg/mL of sodium chloride. The commercial formulation was used as such.

Example 7 - Stabilizing effect on monoclonal antibody formulations.

[00089] Alkyi amine oxide compounds tested include ΙΜ,Ν - Dimethyldecylamine N- oxide (Amine oxide 1, CAS 2605-79-0, Sigma-Aldrich, CMC = 10 mM), N,N - Lauryldimethylamine N-oxide (CAS 1643-20-5, Sigma-Aldrich, CMC = 1 mM), N,N - Dimethyitetradecylamine N-oxide (Amine oxide 3, CAS 3332-27-2, Sigma-A!drich, CMC = 0.14 mM) and N-2-(dimethyinitrory!)ethyldodecanamide (Amine oxide 4, CAS 86321-42-8, Sigma-Aldrich, CMC = 1.8 mM).

[00090] Ail these amine oxide compounds are employed at a concentration equal to 1.5 times their critical micelle concentration.

[00091] The physical stability of each monocionai antibody formulation is evaluated using the following mechanical stress: 0.5 mL of the formulation is shaken on a wheel agitator at a speed of 80 rpm in presence of 2 glass beads of 2 mm-diameter.

[00092] For each monoclonal antibody, three conditions of formulation were tested :

1. The first formulation is the control formulation described in example 1-5.

2. The second formulation is the formulation described in example 1-5 to which polysorbate is added. The same kind of polysorbate as the commercial formulation is added (e.g. for Bevacizumab: polysorbate 20, for Infliximab: polysorbate 80). The final concentration of polysorbate is equal to 1.5 mM.

3. The last formulation is the formulation described in example 1-5 to which an amine oxide compound is added, The concentration of the amine oxide in the formulation is equal to 1.5 times its CMC.

[00093] Monitoring of these experiments is done by visual inspection and light scattering measurements.

[00094] In all the tables: "Clear" means stable , and "Turbid" means unstable. [00095] The table below indicates the visual aspect of each formulation at the end of the stress test.

[00096] Physical stability of the five monoclonal antibody formulations evaluated is drastically increased in presence of amine oxide compou nds.

Example 8 - Stabilizing effect on a fusion protein

[00097] The physical stability of formulations containing a fusion protein was evaluated using the following mechanical stress: 0.5 mL of the formulation was shaken on a wheel agitator at a speed of 80 rpm in presence of 2 glass beads of 2 mm-diameter,

[00098] Two formulations were tested :

1. The first formulation is the control formulation described in example 6,

2. The second formu lation is the control formulation described in example 6 to which amine oxide 2 is added . The concentration of amine oxide 2 in the formulation is 1 , 5 mM which corresponds to 1.5 times its CMC.

[00099] Monitoring of this experiment is done by visual inspection and light scattering measurements.

[000100] The table below indicates the visual aspect of each formu lation at the end of the stress test.

[000101 ] Physical stability of the fusion protein formulation evaluated is drastically increased in presence of a!kyl amine oxide surfactant.

Example 9 - Comparison of the stabilizing effect of different surfactants

[000102] Non-ionic surfactants tested include polysorbate 20 (SI, CAS 9005-64-5, InResa Pharma), polysorbate 80 (S2, CAS 9005-65-6, Sigma-Aldrich), n-Dodecyl β-D- maltoside (S3, CAS 69227-93-6, Sigma-Aldrich), Poiyoxyl 35 hydrogenated castor oil (Ξ4, Cremophor EL, CAS 61791- 12-6, Sigma-Aldrich), Polyoxyethylene- polyoxypropylene block copolymer (S5, Pluronic F-68, CAS 9003- 11-6, Sig ma-Aldrich) .

[000103] Zwitterionic surfactant tested is (Lauryldimethylammonio)acetate (S6, CAS 683- 10-3, Sigma-Aldrich) . [000104] Cationic surfactants tested are Hexadecyltrimethylammonium bromide (S7, CAS 57-09-0, Sigma-Aldrich) and Dodecyltrimethylammonium bromide (S8, CAS 1119-94-4, Sigma-A!drich).

[000105] Oxide surfactants tested are Dimethyldecylphosphine oxide (S9, CAS 2190- 95-6, Sigma-Aldrich) and Dodecylmethyl sulfoxides (S10, CAS 3079-30-9, Sigma- Aldrich).

[000106] Alky! amine oxide compounds tested are N,N - Dimethyldecylamine N-oxide (Amine oxide 1, CAS 2605-79-0, Sigma-Aldrich, CMC = 10 m ), Laury!dimethyiamine N-oxide (Amine oxide 2, CAS 1643-20-5, Sigma-Aldrich, CMC = 1 mM), N,N - Dimethyltetradecy!amine N-oxide (Amine oxide 3, CAS 3332-27-2, Sigma-Aldrich, CMC = 0.14 mM) and N-[2-(dimethylnitroryl)ethyldodecanamide (Amine oxide 4, CAS 86321-42-8, Sigma-Aldrich, CMC = 1.8 mM).

[000107] All theses surfactants are employed at a concentration equal to 1.5 times their critical micelle concentration.

[000108] The physical stability of different formulations containing Bevacizumab is evaluated using the following mechanical stress: 0.5 mL of the formulation is shaken on a wheel agitator at a speed of 80 rpm in presence of 2 glass beads of 2 mm- diameter,

• The first formulation is the control formulation described in example 1.

• The others formulations consist in the control formulation described in example 1 to which a surfactant is added.

[000109] Monitoring of this experiment is done by visual inspection and light scattering measurements.

[000110] The table below indicates the visual aspect of each formulation at the end of the stress test.

Formulation Visual observations

Control formulation Turbid

Control formulation + SI Turbid

Control formulation + S2 Turbid

Control formulation + S3 Turbid

Contro! formulation + S4 Turbid

Control formulation + S5 Tu rbid

Control formulation + Ξ6 Turbid

Control formulation + S7 Turbid

Control formulation + S8 Turbid

Control formulation + S9 Turbid

Control formulation + S10 Turbid

Control formulation + Amine oxide 1 Clear

Control formulation + Amine oxide 2 Clear

Control formulation + Amine oxide 3 Clear

Contro! formulation + Amine oxide 4 Clear

Example 10 - Pharmaceutical formulations

Claims

1. A storage-stable pharmaceutical composition, comprising an aqueous solution of:

(a) At least an antibody-derived therapeutically active protein chosen amongst antibody, nanobody or fusion protein ;

(b) an amount effective to stabilize said antibody-derived therapeutically active protein of at least one lauryldimethylamineoxide and/or of one of its amine oxide analogs.

2. The pharmaceutical composition of claim 1, wherein the analogs of lauryldimethylamineoxide are chosen amongst the amine oxide analogs of formula I :

2 Formula I

wherein

^» m represents an integer comprised in the interval from 0 to 17, 0 < m < 17,

■ n represents an integer comprised in the interval from 0 to 17, 0 < n < 17, ■a represents an integer equal to 0 or 1,

« 9 < m +n < 17,

■If m=0, then a=0

^■F represents a function chosen in the group constituted by the functions amide, ester, carbamate and urea,

» Rl and R2, identical or different, represent alkyl chains comprising from 1 to 4 carbon atoms.

3. The pharmaceutical composition according to claim 2, wherein a = 0 = 0 and the amine o he compounds of formula II:

rmula II

wherein,

^■ n represents an integer comprised in the interval from 9 to 17, 9 < n < 17, ■ l and R2, identical or different, represent alkyl chains comprising from 1 to 4 carbon atoms.

4. The pharmaceutical composition according to any of the previous claims, wherein the concentration of the at least one lauryldimethylamineoxide and/or amine oxide analogs is from 0.01 to lOOmM.

5. The pharmaceutical composition according to any of the previous claims, wherein the concentration of the at least one lauryldimethylamineoxide and/or amine oxide analogs is from 0.1 to lOmM.

6. The pharmaceutical composition according to any of the previous claims, wherein the at least one amine oxide analogs is N,N - Dimethyldecylamine N- oxide (CAS 2605-79-0).

7. The pharmaceutical composition according to any of the previous claims, wherein the at least one amine oxide analogs is N,N - Lauryldimethylamine N- oxide (CAS 1643-20-5).

8. The pharmaceutical composition according to any of the previous claims, wherein the at least one amine oxide analogs is N,N - Dimethyltetradecylamine N-oxide (CAS 3332-27-2).

9. The pharmaceutical composition according to any of the previous claims, wherein the at least one amine oxide analogs is N-2- (dimethylnitroyl)ethyldodecanamide (CAS 86321-42-8).

10. The pharmaceutical composition according to any of the previous claims, further comprising an additional surfactant.

11. The pharmaceutical composition according to any of the previous claims, wherein said composition has a pH that is in the range from 5 to 8.

12. The pharmaceutical composition according to any of the previous claims, wherein said composition has a pH that is in the range from 5.5 to 7.5.

13. The pharmaceutical composition according to any of the previous claims, wherein said composition has an osmolality in the range from about 150 to 800 mOsm/kg.

14. The pharmaceutical composition according to any of the previous claims, wherein said composition has an osmolality in the range from about 200 to 500 mOsm/kg.

15. The pharmaceutical composition according to any of the previous claims, wherein the antibody-derived therapeutically active protein is an antibody.

16. The pharmaceutical composition according to any of the previous claims, wherein the antibody-derived therapeutically active protein is a nanobody.

17. The pharmaceutical composition according to any of the previous claims, wherein the antibody-derived therapeutically active protein is a fusion protein.

18. The pharmaceutical composition according to any of the previous claims, wherein the concentration of the antibody-derived therapeutically active protein is from 1 to 350 mg/mL.

19. A pharmaceutical container, comprising a hermetically sealed vessel and the pharmaceutical composition of claim 1.

20. The pharmaceutical container of claim 19, where in the vessel is a vial, bottle, pre-filled syringe or pre-filled auto-injector.

21. Use of at least one lauryldimethylamineoxide and/or of one of its amine oxide analogs to stabilize a stored, aqueous pharmaceutical formulation of an antibody-derived therapeutically active protein.

22. Use according to claim 21, wherein the at least one amineoxide analog is chosen amongst the amine oxides of formula I:

Formula I

■ wherein

^■ m represents an integer comprised in the interval from 0 to 17, 0 < m <

17,

■n represents an integer comprised in the interval from 0 to 17, 0 < n < 17, ■a represents an integer equal to 0 or 1,

■9 < m +n < 17,

■If m=0, then a=0

■F represents a function chosen in the group constituted by the functions amide, ester, carbamate and urea,

■RI and R2, identical or different, represent alkyl chains comprising from 1 to 4 carbon atoms.

23. A method of providing storage stability to an aqueous pharmaceutical formulation of an antibody-derived therapeutically active protein, comprising admixing under sterile conditions in an aqueous solution an antibody-derived therapeutically active protein, and amount effective to stabilize said antibody- derived therapeutically active protein of at least one lauryldimethylamineoxide and/or of one of its amine oxide analogs.

24. Method according to claim 23, wherein the at least one amine oxide analog is chosen amongst the amine oxides of formula I:

Formula I ^■ wherein

■ m represents an integer comprised in the interval from 0 to 17, 0 ≤ m <

17,

■ n represents an integer comprised in the interval from 0 to 17, 0 < n < 17,

■ a represents an integer equal to 0 or 1,

■ 9 < m +n < 17,

^■ If m = 0, then a = 0

■ Rl and R2, identical or different, represent alkyl chains comprising from 1 to 4 carbon atoms.

25. A method of increasing the shelf like of an aqueous pharmaceutical formulation of an antibody-derived therapeutically active protein, comprising admixing under sterile conditions in an aqueous solution an antibody-derived therapeutically active protein, and amount effective to stabilize said antibody- derived therapeutically active protein of at least one lauryldimethylamineoxide and/or of one of its amine oxide analogs.

26. A method according to claim ¥7- 25, wherein the at least amine oxide analog is chos mula I :

Formula I

wherein

m represents an integer comprised in the interval from 0 to 17, 0 < m < 17,

9 < m +n < 17,

If m = 0, then a = 0