RU2775944C2 - Pharmaceutical composition - Google Patents

Abstract

FIELD: pharmaceutics.

SUBSTANCE: group of inventions relates to the field of pharmaceutical compositions including an antibody or its fragment suitable for lyophilization. A pharmaceutical composition is proposed, including from 80 to 200 mg/ml of an antibody that specifically binds to human FcRn or its antigen-binding fragment, from 7% to 10% wt./vol. of sucrose, a mixture of from 10 mM to 50 mM of histidine and from 180 mM to 250 mM of proline, from 0.01% to 1.0% wt./vol. of polysorbate. The antibody or its antigen-binding fragment include a variable domain of a light chain of SEQ ID NO: 7 and a variable domain of a heavy chain of SEQ ID NO: 8. The composition has pH from 4.0 to 7.5. A method for the production of the specified composition, a lyophilized composition, its recovery method, as well as the use of the pharmaceutical composition or the lyophilized composition for the treatment of inflammatory or autoimmune diseases are also proposed.

EFFECT: inventions provide stable compositions demonstrating increase in aggregates of only 0.2%/month at 30°C and 0.4%/month at 40°C, having short recovery time and low viscosity values.

13 cl, 2 ex, 5 tbl, 13 dwg

Description

The field of technology to which the invention belongs

The present invention relates to the field of pharmaceutical compositions. More specifically, it refers to a pharmaceutical composition, liquid or lyophilized, comprising an antibody, sucrose, an amino acid or mixture of amino acids and a surfactant.

Prerequisites for the creation of the invention

The industrial production of therapeutic antibodies is a difficult task. This requires scaling up from processes developed in the laboratory to produce antibodies in milligram quantities to multi-kilogram quantities. Although antibodies are fairly stable molecules, when stored at high concentrations for some period of time, they can nevertheless suffer from chemical and physical instability. Typical chemical instability can result in deamidation, hydrolysis, oxidation, beta elimination, disulfide exchange, or reduction. Physical instability can lead to denaturation, aggregation or precipitation.

Although liquid formulations can be administered with minimal pre-treatment, they have the disadvantage that they become less stable over time. Water-based liquid compositions are particularly complex; water acts as a reactant or facilitates the transport of reactants, leading to chemical degradation and instability of proteins. Stabilization of proteins in liquid compositions to avoid or minimize aggregation, precipitation, or degradation remains a particular challenge. Aggregation to form an insoluble substance or precipitate is a particular problem. This can cause various problems, such as the formation of aggregates, which can lead to immunological reactions upon administration and/or difficulties in properly administering the pharmaceutical composition, such as blocking the delivery device.

Antibodies can be formulated as freeze-dried, i. lyophilized, solvent-reconstituted form immediately prior to administration. Freeze-dried antibody compositions are generally more stable than aqueous liquid compositions. Lyophilization helps to achieve high concentrations of antibody in the final composition, thus reducing the injection volume as well as the injection time.

In addition, freeze-drying (lyophilization) is the preferred stabilization method for long-term storage of monoclonal antibodies that are otherwise unstable in liquid form. Lyophilization of antibodies at low temperature reduces damage to products and helps maintain molecular integrity. This extends the shelf life, reduces the temperature requirements for transportation, and preserves the chemical and biological properties of the antibodies.

Antibodies, like other proteins, are susceptible to denaturation by lyophilization in the absence of stabilizers. Various stabilizers such as sugars or polyols are typically added to the compositions to protect the antibodies from degradation during lyophilization and storage. The level of stabilization provided by sugars or polyols generally depends on their concentrations. Increasing the sugar/polyol concentration to a certain level can result in reaching the limit of stabilization or even destabilization of the protein during lyophilization, so a certain concentration of stabilizer relative to protein is required for storage stability of the lyophilized antibody (Chang L, et al. J Pharm Sci 2005;94:1445-55). The level of stabilizers used for protection during lyophilization depends on the composition of the preparation, the concentration and physical properties of the stabilizer, and its compatibility with the antibody. The stabilization mechanism in the drying process is such that the stabilizer acts as a substitute for water. The interaction between water and proteins is critical for the conformational stability of proteins. When water is removed during the drying process, stabilizers can form hydrogen bonds with the protein, as water molecules do, thus preserving the natural structure of the protein during lyophilization (Chang L, et al. J Pharm Sci. 2005; 94:1445-55).

While lyophilization is the correct choice to achieve the desired long shelf life for antibodies, there is no simple universal protocol for formulating antibody formulations. Antibodies may become unstable during lyophilization and/or long term storage, aggregates may form upon reconstitution, or lyophilized bars may take too long to reconstitute. A high concentration of stabilizers can affect the physico-chemical properties (ie viscosity) of the final composition.

In view of the foregoing, there remains a need in the art to provide even more improved pharmaceutical formulations of antibodies suitable for lyophilization.

The essence of the invention

The present invention addresses the above problem by providing pharmaceutical compositions comprising an antibody or fragment thereof suitable for freeze-drying (ie, lyophilization). The resulting pharmaceutical compositions may be provided as freeze-dried (ie, lyophilized) compositions that can be reconstituted in a solvent at the time of use, or as reconstituted liquid compositions ready for administration. Recovery in reduced volumes allows you to get a composition with high concentrations of the antibody or its fragment with a low level of aggregation and delivery time.

The following specific embodiments are presented as numbered below:

1. Pharmaceutical composition, including:

a. an antibody or antigen-binding fragment thereof;

b. 1% to 20% w/v sucrose;

c. an amino acid or mixture of amino acids; and

d. surface-active substance.

2. Pharmaceutical composition according to embodiment 1, characterized in that it has a pH of 4.0 to 7.5.

3. The pharmaceutical composition according to Embodiment 1 or Embodiment 2, wherein the antibody or antigen-binding fragment thereof is present at a concentration of 1 to 200 mg/ml, preferably 50 to 150 mg/ml, more preferably 80 to 140 mg/ml .

4. A pharmaceutical composition according to any of the preceding embodiments, wherein the composition comprises 5% to 13% w/v sucrose, preferably 7% to 10% w/v sucrose, more preferably 7% w/v sucrose .

5. A pharmaceutical composition according to any of the preceding embodiments, wherein:

a. the amino acid is histidine or proline; or

b. the mixture of amino acids includes histidine and proline.

6. A pharmaceutical composition according to any of the preceding embodiments, wherein the composition comprises 0.01 to 1.0% w/v, preferably 0.01% to 0.5% w/v of a surfactant.

7. Pharmaceutical composition according to embodiment 6, wherein the surfactant is polysorbate 80.

8. A pharmaceutical composition according to any of the preceding embodiments, wherein the antibody, or antigen-binding fragment thereof, specifically binds to a human FcRn.

9. A pharmaceutical composition according to any of the preceding embodiments, wherein the antibody is a human or humanized antibody.

10. A pharmaceutical composition according to any of the preceding embodiments, wherein the antibody, its antigen binding fragment comprises a light chain variable region as defined in SEQ ID NO: 7 and a heavy chain variable region as defined in SEQ ID NO: 8.

11. A pharmaceutical composition according to any of the preceding embodiments, wherein the composition comprises 1 mg/mL to 200 mg/mL of an antibody or antigen-binding fragment thereof, 10 mM to 50 mM histidine, 10 to 300 mM proline, 1% up to 20% w/v sucrose, 0.01% to 1.0% w/v polysorbate 80, at pH 5.6; preferably the composition comprises 50 mg/ml to 150 mg/ml of antibody or antigen-binding fragment thereof, 20 mM to 40 mM histidine, 50 to 280 mM proline, 5% to 13% w/v sucrose, 0.01 % to 0.5% w/v polysorbate 80, at pH 5.6; more preferably, the composition comprises 80 mg/ml to 140 mg/ml of antibody or antigen-binding fragment thereof, 30 mM histidine, 180 mM proline, 7% w/v sucrose, 0.03% w/v polysorbate 80, at pH 5 .6; and even more preferably, the composition comprises 100 mg/ml of antibody or antigen-binding fragment thereof, 30 mM histidine, 180 mM proline, 7% w/v sucrose, 0.03% w/v polysorbate 80, at pH 5.6.

12. A pharmaceutical composition according to any of the preceding embodiments, wherein the composition further comprises mannitol and/or arginine and/or glycine.

13. A lyophilized pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, sucrose, an amino acid or mixture of amino acids, and optionally a surfactant.

14. Lyophilized composition in accordance with embodiment 13, where the composition comprises from 10% to 40% wt./wt. sucrose, preferably from 20% to 40% wt./wt. sucrose.

15. A lyophilized composition comprising an antibody comprising a light chain variable region as defined in SEQ ID NO: 7 and a heavy chain variable region as defined in SEQ ID NO: 8, and from 10% to 40% w/w. sucrose.

16. A liquid pharmaceutical composition comprising a pharmaceutical composition according to any of embodiments 13-15 and a solvent.

17. A liquid pharmaceutical composition according to Embodiment 16 wherein the solvent is water.

18. A container comprising a lyophilized composition according to any one of embodiments 13-15 or a liquid pharmaceutical composition according to any one of embodiments 16 or 17.

19. A pharmaceutical composition according to any of Embodiments 1-12, a lyophilized composition according to any of Embodiments 13-15, or a liquid pharmaceutical composition according to any of Embodiments 16-17 for use in therapy.

20. A pharmaceutical composition according to any one of embodiments 1-12, a lyophilized composition according to any one of embodiments 13-15, or a liquid pharmaceutical composition according to any one of embodiments 16-17 for use in the treatment of inflammatory or autoimmune diseases, selected from myasthenia gravis, pemphigus vulgaris, optic neuromyelitis, Guillain-Barré syndrome, lupus and thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura (ITP), chronic inflammatory demyelinating polyneuropathy (CIDP), and combinations thereof.

21. A method for treating an inflammatory or autoimmune disease in a mammalian subject, comprising administering a pharmaceutical composition according to any one of Embodiments 1-12, a lyophilized composition according to any one of Embodiments 13-15, or a liquid pharmaceutical composition according to any one of Embodiments implementation 16-17; wherein the inflammatory or autoimmune disease is selected from myasthenia gravis, pemphigus vulgaris, optic neuromyelitis, Guillain-Barré syndrome, lupus and thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura (ITP), chronic inflammatory demyelinating polyneuropathy (CIDP), and combinations thereof.

22. A method for preparing a pharmaceutical composition comprising an anti-FcRn antibody, wherein the method includes the following steps:

(i) obtaining a composition comprising an anti-FcRn antibody; and

(ii) adding 1%-20% w/v sucrose.

23. The method according to Embodiment 22, wherein the pharmaceutical composition obtained in step (ii) is a pharmaceutical composition according to any one of Embodiments 1-12.

24. The method according to Embodiment 22 or Embodiment 23, wherein the method further comprises the step of lyophilizing the pharmaceutical composition obtained in step (ii).

25. Pharmaceutical composition obtained by the method in accordance with embodiment 24.

26. The method of reconstituting a pharmaceutical composition according to any one of embodiments 13, 14, 15, 24, or 25 by adding a solvent, where the solvent is preferably water.

27. Liquid pharmaceutical composition obtained by the method in accordance with embodiment 26.

Brief description of the drawings

Fig. 1 - Recovery time after lyophilization. The recovery time (release time) is shown on the y-axis in minutes for each of the compositions (x-axis) of Table 2.

Fig. 2 - Formation of aggregates, determined using SEC. The total number of aggregates is shown as area % on the y-axis for each of the compositions (x-axis) of Table 2.

Fig. 3 - Formation of HMW aggregates, determined using DLS. Intensities (%) are plotted on the y-axis for each composition (x-axis) of Table 2.

Fig. 4 - Options that differ in charges. The formation of acidic (A) and basic (B) variants are plotted as a function of area % (y-axis) for each composition (x-axis) of Table 2.

Fig. 5 - Viscosity. Viscosity (cP) is shown on the y-axis for each formulation (x-axis) of Table 2.

Fig. 6 - Osmolality. Osmolality (mOsm/kg) is shown on the y-axis for each composition (x-axis) of Table 2.

Fig. 7 - Recovery time after lyophilization. The recovery time (release time) is shown on the y-axis in minutes for each of the compositions (x-axis) of Table 5.

Fig. 8 - Formation of aggregates, determined using SEC. The total number of aggregates as % area is shown on the y-axis for each of the compositions (x-axis) of Table 5.

Fig. 9 - Formation of HMW aggregates, determined using DLS. Masses (%) are plotted on the y-axis for each composition (x-axis) of Table 5.

Fig. 10 - Options that differ in charges. The production of acidic (A) and basic (B) variants is plotted as a function of area % (y-axis) for each formulation (x-axis) of Table 5 containing anti-FcRn antibody at 100 mg/mL.

Fig. 11 - Options that differ in charges. The production of acidic (A) and basic (B) variants is plotted as a function of area % (y-axis) for each composition (x-axis) of Table 5 containing anti-FcRn antibody at 140 mg/mL.

Fig. 12 - Viscosity. Viscosity (cP) is shown on the y-axis for each formulation (x-axis) of Table 5.

Fig. 13 - Osmolality. Osmolality (mOsm/kg) is shown on the y-axis for each composition (x-axis) of Table 5.

Detailed description of the invention

The pharmaceutical composition according to the invention comprises an antibody or an antigen-binding fragment thereof; 1% to 20% w/v sucrose; an amino acid or mixture of amino acids; and a surfactant.

The concentration of the antibody or antigen-binding fragment thereof in the pharmaceutical composition may be from 1 to 200 mg/ml, preferably from 50 to 200 mg/ml, more preferably from 80 to 140 mg/ml.

Preferably, the antibody or antigen-binding fragment contained in the pharmaceutical composition according to the invention specifically binds to a human FcRn.

The term "specifically binds to a human FcRn", "specifically binds to a human FcRn", and equivalents, as used herein, means that an antibody will bind to a human FcRn with sufficient affinity and specificity to achieve a biologically significant effect. The antibody chosen will typically have a binding affinity for a human FcRn, for example, an antibody can bind to a human FcRn with a Kd value between 100 nM and 1 pM. The affinity of antibodies can be determined, for example, using an analysis based on the method of surface plasmon resonance, such as BIAcore analysis; enzyme-linked immunosorbent assay (ELISA); and competitive analyzes (eg RIA). As defined herein, an antibody, or antigen-binding fragment thereof, that specifically binds to a human FcRn can also bind to another molecule, such as a cyno FcRn, or as, for example, as a non-limiting example, in the case of a bispecific antibody.

FcRn is a non-covalent complex of the α chain FcRn membrane protein and β2 microglobulin (β2M). In adult mammals, FcRn plays a key role in maintaining serum antibody levels by acting as a receptor that binds and recycles isotype IgG antibodies. IgG molecules are endocytosed by endothelial cells and, if bound to FcRn, are returned after transcytosis, for example, to the circulation. In contrast, IgG molecules that do not bind to FcRn enter cells and target the lysosomal pathway, where they are cleaved. An IgG1 variant in which His435 is mutated to alanine results in a selective loss of FcRn binding and a significantly reduced serum half-life (Firan et al. 2001, International Immunology 13:993).

An antibody, or antigen-binding fragment thereof, that binds specifically to a human FcRn preferably also neutralizes the human FcRn.

The term "neutralizes", in the context of the present application, refers to an antibody that inhibits or substantially reduces the biological effect of the molecule to which it specifically binds. Therefore, the expression "antibody neutralizes human FcRn" refers to an antibody that specifically binds to a human FcRn and inhibits or substantially reduces its biological effect, for example by blocking FcRn binding to IgG.

The term "antibody" or "antibodies", in the context of this application, refers to monoclonal or polyclonal antibodies and is not limited to recombinant antibodies that are obtained using recombinant technologies known in this field.

Preferably, the antibody contained in the pharmaceutical composition according to the invention is a monoclonal antibody that specifically binds to a human FcRn.

"Antibody" or "antibodies" includes antibodies derived from any species, in particular mammalian, containing two essentially complete heavy and two essentially complete light chains, human antibodies of any isotype, including IgA ₁ , IgA ₂ , IgD, IgG ₁ , IgG _2a , IgG _2b , IgG ₃ , IgG ₄ IgE and IgM, and modified variants thereof, non-human primate antibodies, eg chimpanzee, baboon, rhesus or cynomolgus monkey, rodent eg mouse, rat or rabbit; goat or equine antibodies and their derivatives, either derived from an avian species such as chicken antibodies or from a fish species such as shark antibodies. The term "antibody" or "antibodies" also refers to "chimeric" antibodies in which the first portion of at least one antibody heavy and/or light chain sequence is derived from the first species and the second portion of the antibody heavy and/or light chain sequence is derived from second kind. Chimeric antibodies of interest include "primatized" antibodies comprising antigen-binding variable domain sequences derived from a non-human primate (eg, marmosets such as baboon, rhesus or cynomolgus monkey) and human constant region sequences. "Humanized" antibodies are chimeric antibodies that contain a sequence derived from a non-human antibody. For the most part, humanized antibodies are human antibodies (recipient antibody) in which residues from the hypervariable region of the recipient are replaced by residues from the hypervariable region or complementarity determining region (CDR) of a non-human species (donor antibody) such as a mouse, rat, rabbit, chicken or other from a human primate having the desired specificity, affinity and activity. In most cases, human (recipient) antibody residues are outside the CDR; those. in the frame region (FR) are further replaced by corresponding non-human residues. In addition, humanized antibodies may include residues that are not present in the recipient antibody or in the donor antibody. These modifications are made to further improve the performance of the antibody. Humanization reduces the immunogenicity of non-human antibodies in humans, thus facilitating the use of antibodies to treat human diseases. Humanized antibodies and some other technologies for their production are well known in this field. The term "antibody" or "antibodies" also refers to human antibodies, which can be obtained as an alternative to humanization. For example, transgenic animals (eg, mice) can be generated that are capable, after immunization, of producing the full repertoire of human antibodies in the absence of endogenous mouse antibody production. For example, homozygous deletion of the antibody heavy chain junction region (JH) gene in chimeric and germline mutated mice has been described to result in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin genes to such germline mutated mice will result in the production of human antibodies with specificity for a particular antigen upon immunization of a transgenic animal carrying human germline immunoglobulin genes with said antigen. Technologies for obtaining such transgenic animals and technologies for isolating and producing human antibodies from such transgenic animals are known in the art. Alternatively, in a transgenic animal, eg a mouse, only the immunoglobulin genes encoding the mouse antibody variable regions are replaced by the corresponding human immunoglobulin variable region gene sequences. Mouse germline immunoglobulin genes encoding antibody constant regions remain unchanged. Thus, antibody effector functions in the immune system of the transgenic mouse, and hence B-cell development, are essentially unchanged, which may lead to an improved antibody response upon antigen challenge in vivo. Once the genes encoding the particular antibody of interest are isolated from such transgenic animals, the genes encoding the constant regions can be replaced with human constant region genes to produce a fully human antibody. The term "antibody" or "antibodies", in the context of the present application, also refers to non-glycosylated antibody.

The term "its antigen-binding fragment" or its grammatical variants in the context of the present application refers to an antibody fragment. An antibody fragment comprises at least one immunoglobulin heavy or light chain domain, as is known in the art, and binds to one or more antigens. Examples of antibody fragments according to the invention include Fab, Fab', F(ab') ₂ and Fv and scFv fragments, as well as diabodies, triabodies, tetrabodies, minibodies, domain antibodies (dAbs) such as sdAbs, VHH or camelid antibodies ( e.g. camelids or llamas such as Nanotela™) and VNAR fragments, single chain antibodies, bispecific, trispecific, tetraspecific or multispecific antibodies formed from antibody fragments, or antibodies including, but not limited to, Fab-Fv or Fab-Fv- fv designs. The antibody fragments defined above are known in the art.

The antibody, or antigen-binding fragment thereof, contained in the pharmaceutical composition according to the invention may be a human or humanized antibody, preferably a humanized monoclonal antibody that specifically binds to a human FcRn.

More preferably, the pharmaceutical composition according to the invention comprises (Table 1):

1) an antibody, or its antigen-binding fragment, which

a. includes CDR-H1 having the sequence defined in SEQ ID NO:1; CDR-H2 having the sequence defined in SEQ ID NO:2; CDR-H3 having the sequence defined in SEQ ID NO:3; CDR-L1 having the sequence defined in SEQ ID NO:4; CDR-L2 having the sequence defined in SEQ ID NO:5 and CDR-L3 having the sequence defined in SEQ ID NO:6; or

b. includes a light chain variable region having the sequence defined in SEQ ID NO: 7 and a heavy chain variable region having the sequence defined in SEQ ID NO: 8; or

c. includes a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 7, and a heavy chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 8;

d. includes a light chain variable region having the sequence defined in SEQ ID NO: 7 and a heavy chain having the sequence defined in SEQ ID NO: 11; or

e. includes a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 7, and a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 11; or

2) an antibody that includes a light chain having the sequence defined in SEQ ID NO: 9 and a heavy chain having the sequence defined in SEQ ID NO: 10; or

3) an antibody that includes a light chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 9, and a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 10.

As used herein, complementarity-determining regions ("CDRs") are defined according to Cabot's nomenclature. The Kabat nomenclature is a standard for numbering residues in an antibody and is commonly used to identify CDR regions (Kabat et al., (1991), 5th edition, NIH publication No. 91-3242).

Table 1 Area Symbol and SEQ ID Amino acid sequence CDR-H1
SEQID NO:1 gftfsnygmv CDR-H2
SEQ ID NO:2 yidsdgdntyyrdsvkg CDR-H3
SEQ ID NO:3 gifrpfly CDR-L1
SEQ ID NO:4 kssqslvgasgktyly CDR-L2
SEQ ID NO:5 lvstlds CDR-L3
SEQ ID NO:6 lqgthfpht Light chain variable region
SEQ ID NO:7 DIQMTQSPSS LSASVGDRVT ITCkssqslv gasgktylyW lfQKPGKAPK RLIYlvstld sGiPSRFSGS GSGTEFTLTI SSLQPEDFAT YYClqgthfp HTFGQGTKLE IK Heavy chain variable region
SEQ ID NO:8 EVPLVESGGG LVQPGGSLRL SCAVSgftfs nygmvWVRQA PGKGLEWVAy idsdgdntyy rdsvkgRFTI SRDNAKSSLY LQMNSLRAED TAVYYCttgi vrPFLYWGQG TLVTVS light chain
SEQ ID NO:9 DIQMTQSPSS LSASVGDRVT ITCkssqslv gasgktylyW lfQKPGKAPK RLIYlvstld sGiPSRFSGS GSGTEFTLTI SSLQPEDFAT YYClqgthfp HTFGQGTKLE IKrtvaapsv fifppsdeql ksgtasvvcl lnnfypreak vqwkvdnalq sgnsqesvte qdskdstysl sstltlskad yekhkvyace vthqglsspv tksfnrgec heavy chain
SEQ ID NO:10 EVPLVESGGG LVQPGGSLRL SCAVSgftfs nygmvWVRQA PGKGLEWVAy idsdgdntyy rdsvkgRFTI SRDNAKSSLY LQMNSLRAED TAVYYCttgi vrPFLYWGQG TLVTVSSast kgpsvfplap csrstsesta algclvkdyf pepvtvswns galtsgvhtf pavlqssgly slssvvtvps sslgtktytc nvdhkpsntk vdkrveskyg ppcppcpape flggpsvflf ppkpkdtlmi srtpevtcvv vdvsqedpev qfnwyvdgve vhnaktkpre eqfnstyrvv svltvlhqdw lngkeykckv snkglpssie ktiskakgqp repqvytlpp sqeemtknqv sltclvkgfy psdiavewes ngqpennykt tppvldsdgs fflysrltvd ksrwqegnvf scsvmhealh nhytqkslsl slgk Heavy Chain Fab
SEQ ID NO: 11 EVPLVESGGG LVQPGGSLRL SCAVSgftfs nygmvWVRQA PGKGLEWVAy idsdgdntyy rdsvkgRFTI SRDNAKSSLY LQMNSLRAED TAVYYCttgi vrPFLYWGQG TLVTVSSast kgpsvfplap sskstsggta algclvkdyf pepvtvswns galtsgvhtf pavlqssgly slssvvtvps sslgtqtyic nvnhkpsntk vdkkvepksc

The illustrated antibody, or antigen-binding fragment thereof, is described in more detail in WO2014019727 (incorporated herein by reference).

Antibody molecules can generally be obtained by culturing a host cell containing a vector encoding an antibody sequence under suitable conditions resulting in the expression of a protein comprising DNA encoding an antibody molecule of the present invention and isolating the antibody molecule.

The antibody molecule may include only a heavy or light chain polypeptide, in which case only the sequence encoding the heavy chain or light chain polypeptide should be used to transfect host cells. To obtain products containing both heavy and light chains, the cell line can be transfected with two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector can be used that includes sequences encoding light chain and heavy chain polypeptides.

An antibody, or antigen-binding fragment thereof, which can be produced on an industrial scale, can be obtained by culturing eukaryotic host cells transfected with one or more expression vectors encoding a recombinant antibody fragment. The eukaryotic host cells are preferably mammalian cells, more preferably Chinese hamster ovary (CHO) cells.

Mammalian cells can be cultured in any medium that supports their growth and recombinant protein expression, preferably the medium is a chemically defined medium that is free of animal products such as animal serum and peptone. There are various cell culture media known to those skilled in the art, including various combinations of vitamins, amino acids, hormones, growth factors, ions, buffers, nucleosides, glucose, or an equivalent energy source present at suitable concentrations to allow cell growth and protein production. . Additional components of the cell culture media can be included in the cell culture medium at suitable concentrations at various times during the cell culture cycle, as will be known to those skilled in the art.

The mammalian cell culture can be carried out in any suitable container, such as a shake flask or a bioreactor, which may optionally be operated in a fed-batch mode, depending on the scale of production required. These bioreactors can be either mechanically agitated or air lifted. Various bioreactors are available for large scale production with capacities greater than 1000L - 50000L, preferably from 5000L to 20000L or up to 10000L. Alternatively, smaller scale bioreactors, such as 2 L to 100 L, can also be used to produce an antibody or antibody fragment.

The antibody, or antigen-binding fragment thereof, is typically present in the supernatant of a mammalian host cell culture, typically a CHO cell culture. With respect to CHO culture methods where the protein of interest, such as an antibody or antigen-binding fragment thereof, is secreted in the supernatant, said supernatant is collected by methods known in the art, typically by centrifugation.

Therefore, the method for producing an antibody or antigen-binding fragment thereof includes the step of centrifuging and isolating the supernatant after cell culture and prior to protein purification. In yet another specific embodiment, said centrifugation is a continuous centrifugation. For the avoidance of doubt, supernatant means the liquid above the precipitated cells as a result of cell culture centrifugation.

Alternatively, the host cells are prokaryotic cells, preferably Gram-negative bacteria. More preferably, the host cells are E. coli cells. Prokaryotic host cells for protein expression are well known in the art (Terpe, K. Appl Microbiol Biotechnol 72, 211-222 (2006)). Host cells are recombinant cells that are genetically engineered to produce a protein of interest, such as an antigen-binding fragment of an antibody. Recombinant E. coli host cells can be derived from any suitable E. coli strain, including MC4100, TG1, TG2, DHB4, DH5α, DH1, BL21, K12, XL1Blue, and JM109. One example is E. coli strain W3110 (ATCC 27,325), a widely used host strain for recombinant protein fermentations. Antibody fragments can also be obtained by culturing modified E. coli strains, such as metabolic mutants or protease-deficient E. coli strains.

The antibody fragment is typically present either in the periplasm of the E. coli host cell or in the culture supernatant of the host cells, depending on the nature of the protein, the scale of production, and the E. coli strain used. Methods for targeting proteins to these compartments are well known in the art (Makrides, S.C.; Microbiol Rev 60, 512-538 (1996)). Examples of suitable signal sequences for directing proteins to the E. coli periplasm include the E. coli PhoA, OmpA, OmpT, LamB, and OmpF signal sequences. Proteins can be directed into the supernatant by relying on natural secretory pathways, or by inducing limited outer membrane permeability to induce protein secretion, and examples of this are the use of the pelB leader sequence, Protein A leader sequence, bacteriocin release protein co-expression, mitomycin-inducible protein release of bacteriocins along with the addition of glycine to the culture medium and co-expression of the kil gene to permeabilize membranes. Most preferably, the recombinant protein is expressed in the periplasm of the E. coli host.

Expression of the recombinant protein in E. coli host cells can also be under the control of an inducible system, whereby expression of the recombinant antibody in E. coli is under the control of an inducible promoter. Many inducible promoters suitable for use in E. coli are well known in the art and depending on the promoter used to express the recombinant protein can be induced by various factors such as temperature or the concentration of a particular substance in the nutrient medium. Examples of inducible promoters include the E. coli lac, tac, and trc promoters, which are induced by lactose or the non-hydrolysable lactose analogue isopropyl-b-D-1-thiogalactopyranoside (IPTG), and the phoA, trp, and araBAD promoters, which are induced by phosphate, tryptophan, and L-arabinose, respectively. Expression can be induced, for example, by adding an inducer or by changing the temperature, when the induction is temperature dependent. If induction of expression of the recombinant protein is achieved by adding the inducer to the culture, the inducer can be added in any suitable manner, depending on the fermentation system and the inducer, for example, by one or more additions, or by gradually adding the inducer during feeding. It should be understood that there may be a delay between the addition of an inducer and the actual induction of protein expression, for example, when the inducer is lactose, there may be a delay before induction of protein expression occurs, with any carbon source initially present being used before lactose.

Cultures of E. coli host cells (fermentations) can be grown in any medium that will support E. coli growth and recombinant protein expression. The medium may be any chemically defined medium such as those described in Durany O, et al. (2004). Studies on the expression of recombinant fuculose-1-phosphate aldolase in Escherichia coli. Process Biochem 39, 1677-1684.

The cultivation of E. coli host cells can take place in any suitable container, such as a shake flask or a fermenter, depending on the desired production scale. There are various fermenters for large scale production with capacities ranging from more than 1000 liters to about 100,000 liters. Preferably, fermenters with a capacity of 1000-50000 liters are used, more preferably 1000-25000, 20000, 15000, 12000 or 10000 liters. Smaller scale fermenters with capacities ranging from 0.5 to 1000 liters can also be used.

The fermentation of E. coli can be carried out in any suitable system, eg, continuous, batch or fed-batch, depending on the protein and desired yields. The batch method can be used with additions of small amounts of nutrients or inducers as required. Alternatively, fed-batch cultures can be used and cultures grown in pre-induction batching at maximum specific growth rate can be maintained using the nutrients originally present in the fermenter and one or more nutrient-fed regimens used to control the rate. growth until the completion of fermentation. You can also use the fed-batch method before induction to control the metabolism of E. coli host cells and to achieve greater cell density.

If desired, host cells can be collected from the fermentation medium, for example, host cells can be collected from a sample by centrifugation, filtration or concentration. In this case, the method typically includes the step of centrifuging and isolating the cells prior to protein extraction.

With respect to E. coli fermentation methods where the protein of interest, such as an antibody or antigen-binding fragment of an antibody, is present in the periplasmic space of the host cell, it is necessary to isolate the protein from the host cell. Isolation can be carried out by any suitable method, such as cell lysis using mechanical treatment or pressure, freeze-thaw treatment, osmotic shock, extractants, or heat treatment. Such extraction methods for protein isolation are well known in the art. Therefore, in a particular embodiment, the preparation method includes an additional protein extraction step prior to protein purification.

Other methods for obtaining an antigen-binding fragment of a human antibody in vitro are based on display technologies, such as phage display or ribosome display technology, where recombinant DNA libraries are used that are obtained either at least in part artificially or from the variable gene repertoire ( V) immunoglobulin domains from donors. Phage and ribosome display technologies for generating human antibodies are well known in the art. Human antibodies can also be generated from isolated human B cells that are immunized ex vivo with an antigen of interest and then fused to form hybridomas that can then be screened for the optimal human antibody.

The pharmaceutical composition according to the invention may have a pH of 4.0 to 7.5, preferably 4.0 to 6.0, more preferably 4.5 to 6.0, even more preferably 5.5 to 5.8 or most preferably about 5.6.

The pharmaceutical composition contains a buffering agent to maintain the pH at a constant level. There are many buffering agents used in the field of liquid pharmaceutical compositions such as, but not limited to, citrate, phosphate, lactate, histidine, glutamate, maleate, tartrate or succinate. The preferred type of buffer is usually selected from those that have a pKa value close (pH unit +/- 1) to the preferred pH for optimal protein stability to maintain a high buffering capacity and is associated with the maximum demonstrated stability observed for a particular protein, placed in a number of different types of buffers. Appropriate composition pH ranges are generally selected from those associated with the maximum demonstrated stability observed for a particular protein when placed in a range of pH varying compositions.

The pharmaceutical composition according to the invention comprises, as a buffering agent, an amino acid or a mixture of amino acids, preferably at a concentration of 10 mM to 100 mM, 10 mM to 80 mM, 10 mM to 60 mM, 25 mM to 60 mM, preferably 30 mM to 50 mM, or 30 mM.

Suitable amino acids are arginine, lysine, histidine, methionine, ornithine, isoleucine, leucine, alanine, glycine, glutamic acid or aspartic acid. Preferred is the inclusion of a basic amino acid, ie arginine, lysine and/or histidine. The amino acid may be present in its D and/or L form, but the L form is typical. The amino acid may be present as any suitable salt, for example the hydrochloride such as arginine-HCl.

Preferably the amino acid is histidine, more preferably at a concentration of 30 mM.

The pharmaceutical composition according to the invention may also include a mixture of amino acids, for example a mixture of histidine and proline, histidine, proline and glycine, histidine, proline and arginine, histidine, proline, arginine and glycine. Preferably, the pharmaceutical composition according to the invention comprises histidine and another amino acid, preferably proline, at a concentration of 10 mM to 300 mM, 50 mM to 280 mM, 100 mM to 280 mM, or 180 mM to 250 mM. Preferably, the pharmaceutical composition according to the invention contains 30 mM histidine and 180 mM to 250 mM proline.

The pharmaceutical composition according to the invention further comprises a surfactant. Surfactants available for use in the pharmaceutical composition of the invention include, but are not limited to, non-ionic surfactants, ionic surfactants, and zwitterionic surfactants. Typical surfactants for use with the invention include, but are not limited to, sorbitan fatty acid esters (e.g., sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate), sorbitan trioleate, glycerol fatty acid esters (e.g., glycerol monocaprylate, glycerol monomyristate, glycerol monostearate), сложные эфиры полиглицерина и жирных кислот (например, декаглицерилмоностеарат, декаглицерилдистеарат, декаглицерилмонолинолеат), сложные эфиры полиоксиэтиленсорбитана и жирных кислот (например, полиоксиэтиленсорбитанмонолаурат, полиоксиэтиленсорбитанмоноолеат, полиоксиэтиленсорбитанмоностеарат, полиоксиэтиленсорбитанмонопальмитат, полиоксиэтиленсорбитантриолеат, полиоксиэтиленсорбитан тристеарат), сложные эфиры полиоксиэтиленсорбита и жирных кислот (например, полиоксиэтиленсорбиттетрастеарат, polyoxyethylene sorbitol tetraoleate), polyoxyethylene glycerol fatty acid esters (e.g. polyoxyethylene glyceryl monostearate), complex polyethylene glycol fatty acid esters (e.g. polyethylene glycol distearate), polyoxyethylene alkyl ethers (e.g. polyoxyethylene lauryl ether), polyoxyethylene-polyoxypropylene alkyl ethers (e.g. polyoxyethylene polyoxypropylene glycol, polyoxyethylene-polyoxypropylene propyl ether, polyoxyethylene-polyoxypropylene cetyl ether), polyoxyethylene alkylphenyl ethers ( e.g. polyoxyethylene nonylphenyl ether), polyoxyethylene hydrogenated castor oil {e.g. polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylated beeswax derivatives (e.g. polyoxyethylene sorbitol beeswax), polyoxyethylated lanolin derivatives (e.g. polyoxyethylenelanolin) and polyoxyethylene fatty acid amides (eg polyoxyethylene stearic acid amide); C ₁₀ -C ₁₈ alkyl sulfates (eg sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate), polyoxyethylene C ₁₀ -C ₁₈ alkyl ether sulfate with an average of 2-4 moles of added ethylene oxide units (eg sodium polyoxyethylene lauryl sulfate) and C ₁ -C salts ₁₈ alkyl sulfosuccinate esters (eg sodium lauryl sulfosuccinate ester); and natural surfactants such as lecithin, glycerophospholipid, sphingophospholipids (eg sphingomyelin), and sucrose esters of C ₁₂ -C ₁₈ fatty acids. The composition may include one or more of these surfactants. Preferred surfactants are polyoxyethylene sorbitan fatty acid esters, for example polysorbate 20, 40, 60 or 80. Preferably, the pharmaceutical composition according to the invention comprises polysorbate 80.

The pharmaceutical composition of the invention may include 0.01% to 10% w/v surfactant, 0.01% to 1.0% w/v surfactant, 0.01% to 0, 5% w/v surfactant or 0.03% w/v surfactant such as polysorbates. Preferably, the pharmaceutical composition of the invention comprises 0.03% w/v polysorbate 80.

The pharmaceutical composition in accordance with the present invention includes as a stabilizer (i.e. cryoprotectant) from 1 to 20% wt./about, or from 5% to 13% wt./about, or from 7% to 10% wt./ about, or 7% sucrose.

The present invention also provides a method for preparing a pharmaceutical composition comprising an anti-FcRn antibody or an antigen-binding fragment thereof, which comprises preparing a composition comprising an anti-FcRn antibody, for example, a composition comprising 80 mg/mL to 140 mg/mL of an anti-FcRn antibody or an antigen-binding fragment thereof. an antigen-binding fragment comprising a light chain variable region comprising SEQ ID NO: 7 and a heavy chain variable region comprising SEQ ID NO: 8, 30 mM histidine, 250 mM proline, 0.03% w/v polysorbate 80, at pH 5.6, and adding 1%-25% w/v sucrose.

Preferably, the method includes preparing a composition comprising 80 mg/ml to 140 mg/ml of an anti-FcRn antibody comprising a light chain variable region comprising SEQ ID NO: 7 and a heavy chain variable region comprising SEQ ID NO: 8, 30 mM histidine, 250 mM proline, 0.03% w/v polysorbate 80, at pH 5.6, and adding a composition comprising 30 mM histidine, 0.03% w/v polysorbate 80, from 17.5% to 24.5% w/v sucrose, pH 5.6.

The pharmaceutical compositions described and embodied in the present invention may be in the form of a freeze-dried (ie, lyophilized) pharmaceutical composition.

Therefore, the process for preparing a pharmaceutical composition according to the invention may further include a freeze-drying (ie, lyophilization) step.

Procedures for lyophilizing antibodies are well known in the art (John F. Carpenter and Michael J. Pikal, 1997 Pharm. Res. 14, 969-975); monoclonal antibody products such as SYNAGIS™, REMICADE™, RAPTIVA™, SIMULECT™, XOLAIR™ and HERCEPTIN™ are also currently available and are supplied as lyophilisates. These antibodies are reconstituted to various final concentrations, for example, SIMULECT™ is reconstituted to an antibody concentration of 4 mg/ml, REMICADE™ is reconstituted to a concentration of 10 mg/ml, HERCEPTIN™ to 21 mg/ml, SYNAGIS™ and RAPTIVA™ to 100 mg/ml, and XOLAIR™ up to 125 mg/ml.

Lyophilization methods are discussed in more detail in this application in the Examples section.

The present invention thus provides a lyophilized composition comprising an antibody or antigen-binding fragment thereof, sucrose, an amino acid or mixture of amino acids, and optionally a surfactant. Preferably, the lyophilized composition comprises from 10% to 40% w/w. sucrose, more preferably from 20% to 40% wt./wt. sucrose.

In a preferred embodiment of the present invention, a lyophilized composition comprising from 10% to 40% wt./wt. sucrose also includes an anti-FcRn antibody or an antigen-binding fragment thereof, wherein the antibody or fragment thereof comprises a light chain variable region comprising SEQ ID NO:7 and a heavy chain variable region comprising SEQ ID NO:8.

In another preferred embodiment, the lyophilized composition comprises about 50.8% wt./wt. antibodies or antigen-binding fragment, about 35.5% wt./wt. sucrose, about 3.0% wt./wt. histidine, about 10.5% wt./wt. proline and 0.2% wt./wt. polysorbate 80; wherein the antibody or fragment thereof comprises a light chain variable region comprising SEQ ID NO:7 and a heavy chain variable region comprising SEQ ID NO:8.

Additional excipients for use in the pharmaceutical compositions of the invention include, but are not limited to, viscosity increasing agents, bulking agents, solubilizing agents such as monosaccharides, e.g., fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like. ; disaccharides such as lactose, trehalose, cellobiose and the like; polysaccharides such as raffinose, melesitose, maltodextrins, dextrans, starches, and the like; and polyols such as mannitol, xylitol, maltitol, lactitol, xylitol-sorbitol (glucitol) and the like; polyethylene glycols (eg PEG100, PEG300, PEG600, PEG1500, PEG2000, PEG3000, PEG3350, PEG4000, PEG6000, PEG8000 or PEG20000), polyvinylpyrrolidone, trimethylamine N-oxide, trimethylglycine, or combinations thereof.

Before the freeze-dried (lyophilized) pharmaceutical composition is administered to a patient, it must be reconstituted with a solvent. In the context of the present invention, the preferred solvent is an aqueous solvent, more preferably the aqueous solvent is water.

The volume of solvent used for reconstitution is dictated by the concentration of the antibody or antigen-binding fragment thereof in the resulting liquid pharmaceutical composition. Reconstitution with a lower volume of solvent than the volume before lyophilization produces a composition that is more concentrated than before lyophilization and vice versa.

The present invention therefore provides a process for reconstituting a lyophilized pharmaceutical composition according to the invention by adding a solvent, where the solvent is preferably water.

The reconstituted ratio (volume of pre-lyophilized pharmaceutical composition to solvent used to reconstitute the lyophilized pharmaceutical composition) can vary from 0.5:1 to 1:10. In a preferred embodiment, a ratio of about 1:1 is used so that the resulting volume of reconstituted liquid pharmaceutical composition is about 4.4 ml.

Preferred pharmaceutical compositions for lyophilization and/or reconstituted liquid pharmaceutical compositions according to the invention include:

A. 1 mg/ml to 200 mg/ml of an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising SEQ ID NO:7 and a heavy chain variable region comprising SEQ ID NO:8 from 10 mM to 50 mM histidine, 10 to 300 mM proline, 1% to 20% w/v sucrose, 0.01% to 1.0% w/v polysorbate 80, at pH 5.6;

B. 50 mg/ml to 150 mg/ml of an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising SEQ ID NO:7 and a heavy chain variable region comprising SEQ ID NO:8 from 20 mM to 40 mM histidine, 50 to 280 mM proline, 5% to 13% w/v sucrose, 0.01% to 0.5% w/v polysorbate 80, pH 5.6;

C. 80 mg/ml to 140 mg/ml of an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising SEQ ID NO:7 and a heavy chain variable region comprising SEQ ID NO:8, 30 mM histidine, 250 mM proline, 7% w/v sucrose, 0.03% w/v polysorbate 80 at pH 5.6;

D. 50 mg/ml to 150 mg/ml of an antibody or antigen-binding fragment thereof comprising a light chain variable region comprising SEQ ID NO:7 and a heavy chain variable region comprising SEQ ID NO:8, or a heavy chain comprising SEQ ID NO: 11, 30 mM histidine, 180 mM proline, 7% w/v sucrose, 0.03% w/v polysorbate 80 at pH 5.6;

E. 80 mg/mL to 140 mg/mL, preferably 100 mg/mL, of an antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1 SEQ ID NO:1 , CDR-H2 SEQ ID NO:2 and CDR-H3 SEQ ID NO:3, and the light chain variable region includes CDR-L1 SEQ ID NO:4, CDR-L2 SEQ ID NO:5 and CDR-L3 SEQ ID NO: 6, 30 mM histidine, 180 mM proline, 7% w/v sucrose, 0.03% w/v polysorbate 80 at pH 5.6;

F. 80 mg/ml to 140 mg/ml, preferably 100 mg/ml of an antibody comprising a heavy chain comprising SEQ ID NO: 10 and a light chain comprising SEQ ID NO: 9, 30 mM histidine, 180 mM proline, 7% w/v sucrose, 0.03% w/v polysorbate 80, at pH 5.6, and at least i) or i) and ii) or i), ii) and iii):

i) 100 mM to 250 mM mannitol;

ii) 80 mM to 200 mM L-arginine HCl;

iii) 100 mM to 280 mM glycine;

G. 80 mg/ml to 140 mg/ml, preferably 100 mg/ml of an antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising SEQ ID NO:7 and a heavy chain variable region comprising SEQ ID NO:8, or a heavy chain comprising SEQ ID NO:11, 30 mM histidine, 180 mM proline, 7% w/v sucrose, 0.03% w/v polysorbate 80, at pH 5.6, and at least i) or i) and ii) or i), ii) and iii):

i) 100 mM to 250 mM mannitol;

ii) 80 mM to 200 mM L-arginine HCl;

iii) 100 mM to 280 mM glycine;

H. 80 mg/mL to 140 mg/mL, preferably 100 mg/mL, of an antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1 SEQ ID NO:1 , CDR-H2 SEQ ID NO:2 and CDR-H3 SEQ ID NO:3, and the light chain variable region includes CDR-L1 SEQ ID NO:4, CDR-L2 SEQ ID NO:5 and CDR-L3 SEQ ID NO: 6, 30 mM histidine, 180 mM proline, 7% w/v sucrose, 0.03% w/v polysorbate 80, at pH 5.6, and at least i) or i) and ii) or i) , ii) and iii):

i) 100 mM to 250 mM mannitol;

ii) 80 mM to 200 mM L-arginine HCl;

iii) 100 mM to 280 mM glycine;

I. 80 mg/ml to 140 mg/ml, preferably 100 mg/ml of an antibody comprising a heavy chain comprising SEQ ID NO: 10 and a light chain comprising SEQ ID NO: 9, 30 mM histidine, 180 mM proline, 7% w/v sucrose, 0.03% w/v polysorbate 80, at pH 5.6, and at least i) or i) and ii) or i), ii) and iii):

i) 100 mM to 250 mM mannitol;

ii) 80 mM to 200 mM L-arginine HCl;

iii) 100 mM to 280 mM glycine;

The present invention also provides a container comprising a lyophilized pharmaceutical composition or a reconstituted liquid pharmaceutical composition in accordance with the invention. In particular, the container may be a vial containing the lyophilized pharmaceutical composition to be reconstituted.

The container may be part of a kit consisting of parts, including one or more containers, including lyophilized pharmaceutical compositions in accordance with the invention, suitable solvents for reconstitution of the lyophilized pharmaceutical composition, delivery devices such as syringes, and instructions for use.

Pharmaceutical compositions or liquid pharmaceutical compositions according to the invention are intended for use in therapy.

In a preferred embodiment, the reconstituted liquid pharmaceutical composition for use in therapy comprises 80 mg/mL to 140 mg/mL, preferably 100 mg/mL, of an antibody or antigen-binding fragment thereof, 30 mM histidine, 180 mM proline, 7% w/v sucrose, 0.03% w/v polysorbate 80, at pH 5.6, optionally i) or i) and ii) or i), ii) and iii), where:

i) 100 mM to 250 mM mannitol;

ii) 80 mM to 200 mM L-arginine HCl;

iii) 100 mM to 280 mM glycine;

where the antibody or antigen-binding fragment (as applicable) includes:

1) a light chain variable region comprising SEQ ID NO:7 and a heavy chain variable region comprising SEQ ID NO:8 or a heavy chain comprising SEQ ID NO:11; or

2) a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 7, and a heavy chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 8;

3) a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 7, and a heavy chain having at least 80% identity or similarity, preferably 90 % identity or similarity to the sequence defined in SEQ ID NO: 11;

4) a light chain comprising SEQ ID NO: 9 and a heavy chain comprising SEQ ID NO: 10; or

5) a light chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 9, and a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 10;

6) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1 SEQ ID NO:1, CDR-H2 SEQ ID NO:2 and CDR-H3 SEQ ID NO:3, and a light chain variable region includes CDR-L1 SEQ ID NO:4, CDR-L2 SEQ ID NO:5 and CDR-L3 SEQ ID NO:6.

The pharmaceutical composition or liquid pharmaceutical compositions according to the invention are also intended for use in the treatment of inflammatory or autoimmune diseases selected from myasthenia gravis, pemphigus vulgaris, optic neuromyelitis, Guillain-Barré syndrome, lupus and thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura (ITP) , chronic inflammatory demyelinating polyneuropathy (CIDP), and combinations thereof.

In another preferred embodiment, a reconstituted liquid pharmaceutical composition for use in the treatment of inflammatory or autoimmune diseases selected from myasthenia gravis, pemphigus vulgaris, optic neuromyelitis, Guillain-Barré syndrome, lupus and thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura (ITP), chronic inflammatory demyelinating polyneuropathy (CIDP) and combinations thereof includes from 80 mg/ml to 140 mg/ml, preferably 100 mg/ml of antibody or antigen-binding fragment, 30 mm histidine, 180 mm proline, 7% w/v sucrose, 0.03 % w/v polysorbate 80, at pH 5.6, optionally i) or i) and ii) or i), ii) and iii), where:

i) 100 mM to 250 mM mannitol;

ii) 80 mM to 200 mM L-arginine HCl;

iii) 100 mM to 280 mM glycine;

where the antibody or antigen-binding fragment (as applicable) includes:

1) a light chain variable region comprising SEQ ID NO:7 and a heavy chain variable region comprising SEQ ID NO:8 or a heavy chain comprising SEQ ID NO:11; or

2) a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 7, and a heavy chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 8;

3) a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 7, and a heavy chain having at least 80% identity or similarity, preferably 90 % identity or similarity to the sequence defined in SEQ ID NO: 11;

4) a light chain comprising SEQ ID NO: 9 and a heavy chain comprising SEQ ID NO: 10; or

5) a light chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 9, and a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 10;

6) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1 SEQ ID NO:1, CDR-H2 SEQ ID NO:2 and CDR-H3 SEQ ID NO:3, and a light chain variable region includes CDR-L1 SEQ ID NO:4, CDR-L2 SEQ ID NO:5 and CDR-L3 SEQ ID NO:6.

The invention also provides a method for treating an inflammatory or autoimmune disease in a mammalian subject, comprising administering a pharmaceutical composition or liquid pharmaceutical compositions in accordance with the invention; wherein the inflammatory or autoimmune disease is selected from myasthenia gravis, pemphigus vulgaris, optic neuromyelitis, Guillain-Barré syndrome, lupus and thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura (ITP), chronic inflammatory demyelinating polyneuropathy (CIDP), and combinations thereof.

The mammalian subject may be selected from the group consisting of rodents, cats, dogs, horses, cows, sheep, non-human primates, and humans. Preferably, the mammal is a human.

In yet another preferred embodiment, a method for treating an inflammatory or autoimmune disease in a mammalian subject comprises administering a reconstituted liquid pharmaceutical composition comprising 80 mg/mL to 140 mg/mL, preferably 100 mg/mL of an antibody or antigen-binding fragment thereof, 30 mM histidine, 180 mM proline, 7% w/v sucrose, 0.03% w/v polysorbate 80, at pH 5.6, optional i) or i) and ii) or i), ii) and iii), where:

i) 100 mM to 250 mM mannitol;

ii) 80 mM to 200 mM L-arginine HCl;

ii) 100 mM to 280 mM glycine;

where the antibody or antigen-binding fragment (as applicable) includes:

1) a light chain variable region comprising SEQ ID NO:7 and a heavy chain variable region comprising SEQ ID NO:8 or a heavy chain comprising SEQ ID NO:11; or

2) a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 7, and a heavy chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 8;

3) a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 7, and a heavy chain having at least 80% identity or similarity, preferably 90 % identity or similarity to the sequence defined in SEQ ID NO: 11;

4) a light chain comprising SEQ ID NO: 9 and a heavy chain comprising SEQ ID NO: 10; or

5) a light chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 9, and a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 10;

6) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1 SEQ ID NO:1, CDR-H2 SEQ ID NO:2 and CDR-H3 SEQ ID NO:3, and a light chain variable region includes CDR-L1 SEQ ID NO:4, CDR-L2 SEQ ID NO:5 and CDR-L3 SEQ ID NO:6,

and wherein the inflammatory or autoimmune disease is selected from myasthenia gravis, pemphigus vulgaris, optic neuromyelitis, Guillain-Barré syndrome, lupus and thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura (ITP), chronic inflammatory demyelinating polyneuropathy (CIDP), and combinations thereof.

In addition, the pharmaceutical composition or liquid pharmaceutical compositions according to the invention can be used for the preparation of a medicament for the treatment of inflammatory or autoimmune diseases selected from myasthenia gravis, pemphigus vulgaris, optic neuromyelitis, Guillain-Barré syndrome, lupus and thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura (ITP), chronic inflammatory demyelinating polyneuropathy (CIDP), and combinations thereof.

In another preferred embodiment, the reconstituted liquid pharmaceutical composition can be used to prepare a medicament for the treatment of inflammatory or autoimmune diseases selected from myasthenia gravis, pemphigus vulgaris, neuromyelitis optic nerve, Guillain-Barré syndrome, lupus and thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura (ITP ), chronic inflammatory demyelinating polyneuropathy (CIDP) and combinations thereof, wherein the composition comprises 80 mg/ml to 140 mg/ml, preferably 100 mg/ml of an antibody or antigen-binding fragment thereof, 30 mM histidine, 180 mM proline, 7% wt ./v sucrose, 0.03% w/v polysorbate 80, at pH 5.6, optional i) or i) and ii) or i), ii) and iii), where:

i) 100 mM to 250 mM mannitol;

ii) 80 mM to 200 mM L-arginine HCl;

iii) 100 mM to 280 mM glycine;

where the antibody or antigen-binding fragment (as applicable) includes:

1) a light chain variable region comprising SEQ ID NO:7 and a heavy chain variable region comprising SEQ ID NO:8 or a heavy chain comprising SEQ ID NO:11; or

2) a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 7, and a heavy chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 8;

3) a light chain variable region having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 7, and a heavy chain having at least 80% identity or similarity, preferably 90 % identity or similarity to the sequence defined in SEQ ID NO: 11;

4) a light chain comprising SEQ ID NO: 9 and a heavy chain comprising SEQ ID NO: 10; or

5) a light chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 9, and a heavy chain having at least 80% identity or similarity, preferably 90% identity or similarity to the sequence defined in SEQ ID NO: 10;

6) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR-H1 SEQ ID NO:1, CDR-H2 SEQ ID NO:2 and CDR-H3 SEQ ID NO:3, and a light chain variable region includes CDR-L1 SEQ ID NO:4, CDR-L2 SEQ ID NO:5 and CDR-L3 SEQ ID NO:6.

The reconstituted liquid pharmaceutical compositions according to the invention are administered in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" refers to the amount of a therapeutic agent (i.e., antibody) required to treat, alleviate, or prevent a particular disease, disorder, or condition, or to produce a detectable therapeutic, pharmacological, or prophylactic effect. For any antibody or antigen-binding fragments thereof, a therapeutically effective amount can be initially determined either in cell culture assays or in animal models, typically rodents, rabbits, dogs, pigs, or primates. The animal model can also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes of administration for humans.

The exact therapeutically effective amount for a human subject will depend on the severity of the disease, the general health of the subject, the subject's age, body weight and sex, diet, time and frequency of administration, drug(s) used in the combination, response sensitivity, and resistance/response. for therapy. This amount can be determined by routine experimentation and is subject to the judgment of the attending physician. Typically, a therapeutically effective amount of an antibody will be 0.01 mg/kg to 500 mg/kg, eg 0.1 mg/kg to 200 mg/kg, eg 100 mg/kg.

For the treatment of the above diseases and/or disorders, the appropriate dosage will vary depending on, for example, the particular antibody used, the subject receiving the treatment, the route of administration, and the nature and severity of the condition being treated. In a specific embodiment, the reconstituted liquid pharmaceutical composition of the invention is administered intravenously or subcutaneously. When administered via intravenous injection, the composition may be administered as a bolus injection or as a continuous infusion. The reconstituted liquid pharmaceutical composition according to any of the embodiments of the invention can also be administered by intramuscular injection. The reconstituted liquid pharmaceutical composition may be administered using a syringe, an injection device such as a syringe tube, a needleless device, an implant, and a patch.

In one specific embodiment of the present invention, a dose of 400 mg is achieved by administering 4 ml of a 1:1 reconstituted liquid pharmaceutical composition containing 100 mg/ml of an anti-FcRn antibody or antigen-binding fragment thereof.

The liquid pharmaceutical composition of the invention is suitably administered to a patient at a single time or using a series of injections and may be administered to a patient at any time after diagnosis; it can be administered as the sole treatment or in combination with other drugs or therapies useful in the treatment of the conditions described above.

The antibody, or antigen-binding fragment thereof, may be the sole active ingredient in the liquid pharmaceutical composition. Alternatively, the antibody, or antigen-binding fragment thereof, may be administered in combination, eg, simultaneously, sequentially, or separately, with one or more other therapeutically active ingredients. Active ingredient, as the term is used in this application, refers to an ingredient with a pharmacological effect, such as a therapeutic effect, at an appropriate dose. In some embodiments, the antibody, or antigen-binding fragment thereof, may be present in the liquid pharmaceutical composition along with other active ingredients, including other antibodies or non-antibody ingredients. When the antibody in the liquid pharmaceutical composition according to the invention is an anti-FcRn antibody, an additional active ingredient may be administered to the subject to treat an inflammatory or autoimmune disease. In one embodiment, the subject is administered, simultaneously or sequentially (before and/or after), other ingredients that are antibodies or are other than antibodies, such as steroids or other drug molecules, in particular drug molecules whose half-life is does not depend on FcRn binding.

Hereinafter the invention will be described in more detail by way of examples with reference to the embodiments illustrated in the accompanying drawings.

EXAMPLES

Abbreviations

DLS (dynamic light scattering); Arg(L-arginine); iCE (Capillary Electrophoresis with Imaging); Mal (maltitol); Pro (L-proline); PS80 (polysorbate 80); Raf (raffinose); SE-UPLC (Size-Exclusion Ultra-Performance Liquid Chromatography); Sor (sorbitol); Suc (sucrose); Gly (glycine); Man (mannitol); Met (L-methionine); Tre (trehalose).

materials

D-(-)-sorbitol (VWR GPR Rectopur™); Polysorbate 80 (Tween 80™, Merck™); 140 mg/ml anti-FcRn antibody described in WO2014019727 (SEQ ID NOS: 9 and 10) in 30 mM histidine, 250 mM proline, 0.03% PS80, pH5.6 (UCB Celltech ltd); mannitol (Roquette™); all of the following are from (Sigma™): D-(+)-raffinose pentahydrate, L-arginine monohydrochloride, L-histidine, L-proline, maltitol, glycine, sucrose, L-methionine, and trehalose dehydrate.

Example 1

Preparation of the lyophilized composition

Compositions to be lyophilized comprising 100 mg/ml and 80 mg/ml of the anti-FcRn antibody described in WO2014019727 (SEQ ID NOs: 9 and 10) were prepared by adding to a composition comprising 140 mg/ml of said antibody in 30 mM histidine , pH 5.6, 250 mm proline, 0.03% PS80 (reference composition), solutions A-I, as indicated in Table 2 below.

table 2 Sample Conc. (mg/ml) Composition Conc. (mg/ml) Composition Initial Addendum (100:40) ultimate A N/A 30 mM histidine pH 5.6, 0.03% PS80, 17.5% sucrose 100 30 mM histidine pH 5.6, 180 mM proline, 0.03% PS80, 5% sucrose 100-Suc5 B 30 mM histidine pH 5.6, 0.03% PS80, 24.5% sucrose 30 mM histidine pH 5.6, 180 mM proline, 0.03% PS80, 7% sucrose 100-Suc7 C 30 mM histidine pH 5.6, 0.03% PS80, 14% sorbitol 30 mM histidine pH5.6, 180 mM proline, 0.03% PS80, 4% sorbitol 100-Sor4 D 30 mM histidine pH5.6, 0.03% PS80, 24.5% maltitol 30 mM histidine pH 5.6, 180 mM proline, 0.03% PS80, 7% maltitol 100-Mal7 E 30 mM histidine pH 5.6, 0.03% PS80, 35% raffinose 30 mM histidine pH5.6, 180 mM proline, 0.03% PS80, 10% raffinose 100-Raf10 F 30 mM histidine pH 5.6, 0.03% PS80, 630 mM L-arginine 30 mM histidine pH5.6, 180 mM proline, 0.03% PS80, 180 mM L-arginine 100-Arg180 G 30 mM histidine pH 5.6, 0.03% PS80, 250 mM proline 30 mM histidine pH 5.6, 250 mM proline, 0.03% PS80 100-Pro250 Addendum (80:60) ultimate H N/A 30 mM histidine pH 5.6, 0.03% PS80, 16.5% sucrose 80 30 mM histidine pH5.6, 180 mM proline, 0.03% PS80, 7% sucrose 80-Suc7 I 30 mM histidine pH5.6, 0.03% PS80, 21% sucrose 30 mM histidine pH5.6, 180 mM proline, 0.03% PS80, 9% sucrose 80-Suc9

Approximately 3 ml vials (Schott Topline Fiolax™ clear) were filled with 1 ml of the composition to be lyophilized. Lyophilization was performed using a Martin-Christ Epsilon 2-6D™ lyophilizer. The lyophilization cycle consisted of freezing at -40°C, normalization at -10°C, sublimation at -20°C and secondary drying at 25°C (Table 3). At the end of the lyophilization cycle, the vials were filled with approximately 400 mbar of N2 and sealed with single stoppers (Westar™, West Pharmaceuticals™). The lyophilizates were stored at 2-8° C. until analysis or stability testing.

Table 3 stage t (hour) linear
change (°C/min) holding
(min) T (°C) p (mbar) one 0.00 0 5 1000 2 1.50 0.50 -40 1000 3 2.50 60 -40 1000 four 3.50 0.50 -ten 1000 5 5.50 120 -ten 1000 6 6.50 0.50 -40 1000 7 8.50 120 -40 1000 eight 9.00 thirty -40 0.28 9 9.67 0.50 -twenty 0.28 ten 33.67 1440 -twenty 0.28 eleven 35.17 0.50 25 0.28 12 41.17 360 25 0.28 13 41.50 1.00 5 0.28 fourteen 50.00 0 5 0.28

The stability tests carried out in these experiments consisted of an accelerated stability test for 3 and 6 months at 30° C. and a stress stability test for 4 weeks and 3 months at 40° C.).

1. Recovery

The lyophilizates were reconstituted using 0.9 ml Milli-Q water to a concentration of 80 or 100 mg/ml, respectively. The time for complete dissolution of the briquette to obtain a clear solution (with a possible small amount of foam around the edges on top) was recorded. The appearance of the lyophilizate and reconstituted solution was evaluated visually.

The recovery time is shown in Fig. 1. Reconstitution time is shown on the y-axis in minutes for each of the compositions listed in Table 2. Measurements were made on the composition reconstituted immediately after lyophilization (no storage; t0); after reconstitution of the lyophilized pharmaceutical composition after storage for 3 months at 30°C (t3m, 30°C); after 6 months at 30°C (t6m, 30°C); after 4 weeks at 40°C (t4n, 40°C) and after 3 months at 40°C (t3m, 40°C).

The recovery time was within 10 minutes for the compositions containing sucrose and for the composition with 180 mm L-arginine (Fig. 1). The formulation containing 80 mg/ml of anti-FcRn antibody recovered substantially faster than formulations containing 100 mg/ml. Formulations comprising 100 mg/ml anti-FcRn antibody in 7% sucrose or 180 mM L-arginine showed an acceptable recovery time of less than 10 minutes at all time points in the stability tests.

Aggregation

Anti-FcRn antibody concentration was determined by UV absorbance at 280 nm, undiluted, using a SoloVPE™ (CE Technologies™) extension connected to a Cary50™ UV spectrophotometer (Varian™), or after diluting in water to 1 mg/mL with using a plate reader Spectramax™ M5 (Molecular Devices ^TM ), ε=1.34ml.mg ^-1 .cm ^-1 .

Aggregated protein was quantified by size-exclusion UPLC (SE-UPLC) using two Acquity™ BEH200 SEC (Waters) 1.7 µm, 4.6×150 mm columns in series and running buffer containing 0.1 M Na-phosphate, 0. 3M NaCl, pH7.0 (0.3ml/min). Detection and quantification was carried out using fluorescence analysis (ex. 280 nm, em. 345 nm).

Protein aggregation was also assessed by dynamic light scattering (DLS) using a DynaPro Tablet Reader™ (Wyatt™). Aliquots of samples diluted to 10 mg/ml in 30 mM histidine pH 5.6 were measured (10 samples of 5 sec). Data was analyzed using Dynamics™ 7.1.4 regularization suitable for multimodal particle distribution (Wyatt™).

The data is shown in Fig. 2 as the total percentage of aggregates, determined using SE UPLC, for each composition. A reference composition (140 mg/ml anti-FcRn antibody in 30 mM histidine, pH 5.6, 250 mM proline, 0.03% PS80; non-lyophilized) was also tested (indicated as Ref in FIG. 2). Measurements were made on the composition reconstituted immediately after lyophilization (no storage; t0); after reconstitution of lyophilized pharmaceutical compositions after storage for 3 months at 30°C (t3m, 30°C); after 6 months at 30°C (t6m, 30°C); after 4 weeks at 40°C (t4n, 40°C) and after 3 months at 40°C (t3m, 40°C).

With the exception of the proline composition (100-Pro250), all compositions showed improved aggregation stability compared to the reference composition (FIG. 2). In particular, compositions containing ≥7% sucrose showed good aggregation stability, both in terms of absolute aggregation in each stability test and in aggregation rate in tests performed at 30°C and 40°C. A formulation containing 100mM/mL anti-FcRn antibody in 30mM histidine pH 5.6, 180mM proline, 7% sucrose, 0.03% PS80 showed an increase in aggregates of only 0.2%/month at 30°C and 0 .4%/month at 40°C (Table 4).

Table 4 Agr. (% area) Aggregate speed
(% area/month) _t0 t _3m30°C t _6m30°C t _4h40°C t _3m40°C 30°C 40°C Ref 3.7 7.0 7.6 8.0 13.5 0.9 3.8 100-Suc5 3.6 4.6 4.8 4.6 5.4 0.3 0.8 100-Suc7 3.6 4.3 4.4 4.2 4.8 0.2 0.5 100-Sor4 3.6 4.9 5.4 5.3 6.9 0.4 1.4 100-Mal7 3.6 4.2 4.2 4.1 4.7 0.1 0.5 100-Raf10 3.6 4.5 4.7 4.5 5.2 0.2 0.7 100-Arg180 3.5 4.6 4.9 4.6 5.6 0.3 0.9 100-Pro250 3.9 7.1 8.3 7.4 11.1 1.0 3.1 80-Suc7 3.5 4.0 4.0 3.9 4.3 0.1 0.3 80-Suc9 3.5 3.8 3.7 3.8 4.1 0.1 0.2

Maltitol also showed acceptable aggregation, comparable to that seen with ≥7% sucrose. Although arginine and maltitol performed quite well in terms of recovery time and aggregation, respectively, they did not provide acceptable results in both tests, in contrast to ≥7% sucrose, which provided a short recovery time, low aggregation and formation rate.

Aggregates that were not detected by the SE UPLC method were examined using DLS. Percentage of aggregates, determined by DSL using percent size distribution intensity, is shown for each of the lyophilized compositions, before lyophilization and for compositions reconstituted immediately after lyophilization (no storage; t0); after reconstitution of lyophilized pharmaceutical compositions after storage for 3 months at 30°C (t3m, 30°C); after 6 months at 30°C (t6m, 30°C); after 4 weeks at 40°C (t4n, 40°C) and after 3 months at 40°C (t3m, 40°C).

A reference composition (140 mg/ml anti-FcRn antibody in 30 mM histidine, pH5.6, 250 mM proline, 0.03% PS80; non-lyophilized) was also tested. Neither the reference (Ref) nor the lyophilized formulations showed any significant amount of HMW aggregates. No significant change in large or HMW aggregates affecting stability was observed using DLS (FIG. 3) because the measured intensity deviations are not significant and depend on the measurement procedure.

Variants with different charges

Variants differing in charge were determined by imaging capillary electrophoresis (iCEwith iCE3 (Protein Simple™). Measurements were made for compositions reconstituted immediately after lyophilization (no storage; t0); after reconstitution of lyophilized pharmaceutical compositions after storage for 3 months at 30°C. C (t3m, 30°C); after 6 months at 30°C (t6m, 30°C); after 4 weeks at 40°C (t4n, 40°C) and after 3 months at 40°C (t3m, 40 °C).

An increase in acid components (FIG. 4A) was observed for the reference composition (140 mg/ml anti-FcRn antibody in 30 mM histidine, pH5.6, 250 mM proline, 0.03% PS80; unlyophilized). The formulation containing 250 mM proline showed a decrease in acidic components, but it also showed a significant increase in basic components (FIG. 4B). The composition containing 4% sorbitol also showed a marked increase in the main components. The rest of the lyophilized formulations showed only a slight increase in base components and no significant change in acid components (FIGS. 4A and 4B). Compositions containing ≥7% sucrose did not show any significant change in charge variants.

Viscosity

The viscosity of the reconstituted product was measured using a ViscoPro2000™ viscometer (Cambridge Viscosity). Measurements were made for compositions reconstituted immediately after lyophilization (without storage) at 22±1°C.

Viscosity increased with increasing sugar/sugar alcohol concentration or with increasing sugar/sugar alcohol molecular weight (FIG. 5). The viscosities for the 80 and 100 mg/mL formulation in the sucrose lyophilized formulation were acceptable in both cases. A formulation comprising 100 mg/ml anti-FcRn antibody in combination with 180 mM L-arginine also showed acceptable viscosity. Arginine is a recognized excipient that is commonly added to pharmaceutical compositions to reduce viscosity. Its effect is comparable to a composition containing sucrose.

Osmolality

The osmolality of the reconstituted compositions was measured using a Vapro™ 5520 steam osmometer (Wescor™). Measurements were made for compositions reconstituted immediately after lyophilization (no storage).

Most of the compositions showed an osmolality of about 500 mOsm/kg (Fig. 6). The formulation containing 100mg/ml anti-FcRn antibody in formulation with 180mM L-arginine showed the highest osmolality (570mOsm/kg), while the formulation containing 100mg/ml anti-FcRn antibody in formulation with 250mM proline showed the lowest osmolality (330 mOsm/kg). The composition including sucrose showed acceptable osmolality.

Example 2

Compositions to be lyophilized (Table 5), with the exception of the reference composition, were prepared from 60 mg/ml anti-FcRn antibody in 30 mM histidine, 250 mM sorbitol, pH 5.6, by buffer exchange using Amicon™ Ultra-15 30K ( Millipore™) filter centrifuge/concentrators for volumes up to 15 ml, with at least 4 dilution/concentration cycles using a total diavolume of at least 7 times the volume of the original sample, to obtain final compositions in accordance with Table 5 at pH 5.6. Polysorbate 20 (PS20) was added after buffer exchange from a filtered 3% PS20 stock solution to a final concentration of 0.03%. Composition B of Example 1 (100 mg/ml anti-FcRn antibody, 7% sucrose, 30 mM histidine, 180 mM proline and 0.03% PS80, pH 5.6) was chosen as the reference composition for the experiments of Example 2.

Vials (3 ml; Topline Fiolac clear, Schott™) were filled with 1 ml of formulation containing 100 mg/mL or 140 mg/mL anti-FcRn antibody (asterisk (*) indicates formulations tested at both concentrations of 100 mg/mL and 140 mg /ml) (Table 5). Lyophilization was carried out as in Example 1.

Table 5 Composition % w/v mm % w/v mm %
wt./about mm reference sucrose 7 204 L-proline 180 PS80 0.03 histidine thirty Suc7 7 204 N/A N/A PS20 0.03 histidine thirty Suc10 ^* ten 292 N/A N/A Suc13 13 380 N/A N/A Suc5Man4 5 146 Mannitol four 220 Suc6Man3 ^* 6 175 3 165 Suc7Man2 7 204 2 110 Suc5Arg4 5 146 L-arginine HCl four 190 Suc6Arg3 ^* 6 175 3 142 Suc7Arg2 7 204 2 95 Suc5Gly2 5 146 Glycine 2 266 Suc6Gly1.5 ^* 6 175 1.5 200 Suc7Gly1 7 204 one 133 Suc5Met3 5 146 L-methio-nin 3 201 Suc6Met2.25 ^* 6 175 2.25 151 Suc7Met1.5 7 204 1.5 100 Tre10 ^* Tre-halose (dihydrate) ten 264 N/A N/A

Recovery

Recovery was carried out as in Example 1. Measurements were made for compositions reconstituted immediately after lyophilization (no storage; t0); after reconstitution of lyophilized pharmaceutical compositions after storage for 3 months at 30°C (t3m, 30°C); after 6 months at 30°C (t6m, 30°C); after 4 weeks at 40°C (t4n, 40°C) and after 3 months at 40°C (t3m, 40°C). Relatively fast recovery was achieved for sucrose, sucrose-glycine and sucrose-arginine formulations at both 100 mg/ml and 140 mg/ml anti-FcRn antibody (FIG. 7A, FIG. 7B). The sucrose-mannitol formulations did not achieve a relatively fast recovery, while the methionine and trehalose formulations required a long recovery time (see y-axis difference in Fig. 7A). The difference between the 10% sucrose and 10% trehalose formulations is surprising and suggests a specific reconstitution interaction between sucrose and the anti-FcRn antibody. The negative effect of methionine on recovery could be due to a change in the structure of the lyophilized cake, but also due to molecular changes in the anti-FcRn antibody (more pronounced in the aggregation assay described below).

Aggregation

Aggregation was examined according to the method of Example 1. Measurements were made on compositions before lyophilization, on compositions reconstituted immediately after lyophilization (no storage; t0); after reconstitution of lyophilized pharmaceutical compositions after storage for 3 months at 30°C (t3m, 30°C); after 6 months at 30°C (t6m, 30°C); after 4 weeks at 40°C (t4n, 40°C) and after 3 months at 40°C (t3m, 40°C). All compositions, except for the methionine-containing composition, at both concentrations (100 mg/ml and 140 mg/ml) of anti-FcRn antibodies showed good stability against aggregation (Fig. 8A and 8B). Compositions showing the highest stability against aggregation included compositions with 10% and 13% sucrose, compositions with 6% sucrose-3% mannitol and 7% sucrose-2% mannitol, all arginine-containing compositions, as well as a reference composition (containing 7 % sucrose and proline). The levels of aggregation for the composition containing 140 mg/ml anti-FcRn antibody were the same as for the composition containing 100 mg/ml. The methionine-containing compositions showed a high degree of aggregate formation at both concentrations of the anti-FcRn antibody.

High molecular weight (HMW) aggregates were also investigated. The percentage of aggregates, determined by DSL using the percentage mass particle size distribution, is shown for each of the lyophilized compositions before lyophilization and for the compositions reconstituted immediately after lyophilization (no storage; t0); after reconstitution of lyophilized pharmaceutical compositions after storage for 3 months at 30°C (t3m, 30°C); after 6 months at 30°C (t6m, 30°C); after 4 weeks at 40°C (t4n, 40°C) and after 3 months at 40°C (t3m, 40°C). The results are shown in FIG. 9A and 9B. With the exception of methionine-containing compositions, no significant increase in high molecular weight (HMW) aggregates, as measured by DLS, was observed. The increase in HMW aggregates for methionine-containing formulations is in clear agreement with the SE-UPLC data. Aggregation appears to be induced by heat stress rather than lyophilization.

Variants with different charges

Analysis of charge variants was carried out as in Example 1. Measurements were made on pharmaceutical compositions before lyophilization, on compositions reconstituted immediately after lyophilization (no storage; t0); after reconstitution of lyophilized pharmaceutical compositions after storage for 3 months at 30°C (t3m, 30°C); after 6 months at 30°C (t6m, 30°C); after 4 weeks at 40°C (t4n, 40°C) and after 3 months at 40°C (t3m, 40°C). For compositions containing 100 mg/ml anti-FcRn antibody, a decrease in acidic components and an increase in basic components was observed for methionine-containing compositions after storage at 30°C and 40°C (FIGS. 10A and 10B). Similar behavior was confirmed for compositions containing 140 mg/ml anti-FcRn antibody and methionine (FIGS. 11A and 11B). This suggests thermally-induced chemical changes in the anti-FcRn antibody due to the presence of methionine, which may also be responsible for the aggregation behavior of the anti-FcRn antibody. For all other compositions, no significant changes in charge variants were observed.

Viscosity

The viscosity of the reconstituted product was measured using a ViscoPro2000™ viscometer (Cambridge Viscosity™). The measurements were carried out on the composition reconstituted immediately after lyophilization (without storage). The viscosity for the arginine-containing formulations was very low (<6 cps), even at 140 mg/ml (FIG. 12). For all other compositions, the viscosity was greater than >8 cps.

Osmolality

The osmolality of the reconstituted compositions was measured using a Vapro™ 5520 steam osmometer (Wescor™). The measurements were carried out on the composition reconstituted immediately after lyophilization (without storage). All compositions with good stability and recovery characteristics, such as those containing ≥7% sucrose, showed acceptable osmolality. It is worth noting that the methionine-containing compositions showed very low osmolality.

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Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

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Claims (21)

1. Pharmaceutical composition for the treatment of inflammatory or autoimmune diseases selected from myasthenia gravis, pemphigus vulgaris, optic neuromyelitis, Guillain-Barré syndrome, lupus and thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura (ITP), chronic inflammatory demyelinating polyneuropathy (CIDP) and their combinations including: a. an antibody or antigen-binding fragment thereof, comprising a light chain variable region as defined in SEQ ID NO: 7 and a heavy chain variable region as defined in SEQ ID NO: 8, wherein the antibody or antigen-binding fragment thereof is present at a concentration of 80 to 200 mg/mL ; b. 7% to 10% w/v sucrose; c. a mixture of 10 mM to 50 mM histidine and 180 mM to 250 mM proline; d. from 0.01% to 1.0% w/v of a polysorbate surfactant, and e. having a pH of 4.0 to 7.5. 2. The pharmaceutical composition according to claim 1, wherein the antibody or antigen-binding fragment thereof is present at a concentration of 80 to 150 mg/ml, more preferably 80 to 140 mg/ml. 3. A pharmaceutical composition according to any one of the preceding claims, wherein the composition comprises 7% w/v sucrose. 4. A pharmaceutical composition according to any one of the preceding claims, wherein the composition comprises 0.01% to 0.5% w/v of a polysorbate surfactant. 5. The pharmaceutical composition of claim 4, wherein the polysorbate surfactant is polysorbate 80. 6. A pharmaceutical composition according to any one of the preceding claims, wherein the antibody is a human or humanized antibody. 7. A pharmaceutical composition according to any of the preceding claims, wherein the composition comprises 80 mg/ml to 200 mg/ml of an antibody or antigen-binding fragment thereof, 10 mM to 50 mM histidine, 180 to 250 mM proline, 7% to 10% w/v sucrose, 0.01% to 1.0% w/v polysorbate 80, at pH 5.6; preferably the composition comprises 80 mg/ml to 150 mg/ml of antibody or antigen-binding fragment thereof, 20 mM to 40 mM histidine, 180 to 250 mM proline, 7% to 10% w/v sucrose, 0.01 % to 0.5% w/v polysorbate 80, at pH 5.6; more preferably, the composition comprises 80 mg/ml to 140 mg/ml of antibody or antigen-binding fragment thereof, 30 mM histidine, 180 mM proline, 7% w/v sucrose, 0.03% w/v polysorbate 80, at pH 5.6; and even more preferably, the composition comprises 100 mg/ml of antibody or antigen-binding fragment thereof, 30 mM histidine, 180 mM proline, 7% w/v sucrose, 0.03% w/v polysorbate 80, at pH 5.6. 8. A pharmaceutical composition according to any of the preceding claims, wherein the composition further comprises mannitol and/or arginine and/or glycine. 9. Lyophilized composition for the treatment of inflammatory or autoimmune diseases selected from myasthenia gravis, pemphigus vulgaris, optic neuromyelitis, Guillain-Barré syndrome, lupus and thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura (ITP), chronic inflammatory demyelinating polyneuropathy (CIDP) and their combinations, including an antibody or its antigen-binding fragment, sucrose, a mixture of histidine and proline, and a surfactant, which is a polysorbate, where the composition includes from 20% to 40% wt./wt. sucrose, and wherein the antibody or antigen-binding fragment thereof comprises the light chain variable region as defined in SEQ ID NO: 7 and the heavy chain variable region as defined in SEQ ID NO: 8, and wherein, prior to lyophilization, the antibody or antigen-binding fragment thereof has concentration from 80 to 200 mg/ml; contains from 10 mM to 50 mM histidine, from 180 mM to 250 mM proline; and, if present, from 0.01% to 1.0% w/v surfactant. 10. The use of a pharmaceutical composition according to any one of paragraphs. 1-8 or a lyophilized composition according to claim 9 for the treatment of inflammatory or autoimmune diseases selected from myasthenia gravis, pemphigus vulgaris, optic neuromyelitis, Guillain-Barré syndrome, lupus and thrombotic thrombocytopenic purpura, idiopathic thrombocytopenic purpura (ITP), chronic inflammatory demyelinating polyneuropathy (CIDP) and their combinations. 11. The method of obtaining a pharmaceutical composition according to any one of paragraphs. 1-8, where the method includes the following steps: (i) preparing a composition comprising an antibody or antigen-binding fragment thereof, a mixture of 10 mM to 50 mM histidine and 180 mM to 250 mM proline, and 0.01% to 1.0% w/v of a surfactant, which is a polysorbate; and (ii) adding 7%-10% w/v sucrose, wherein the pharmaceutical composition contains an antibody or an antigen-binding fragment thereof, comprising a light chain variable region as defined in SEQ ID NO: 7 and a heavy chain variable region as defined in SEQ ID NO: 8, wherein the antibody or antigen-binding fragment thereof has a concentration of 80 to 200 mg/ml. 12. The method of claim 10, wherein the method further comprises the step of lyophilizing the pharmaceutical composition obtained in step (ii). 13. The method of restoring the composition according to claim 9, including the addition of a solvent, where the solvent is preferably water.

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